Enzyme stalling method

ABSTRACT

The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.

FIELD OF THE INVENTION

The invention relates to new methods of moving helicases past spacers onpolynucleotides and controlling the loading of helicases onpolynucleotides. The invention also relates to new methods ofcharacterising target polynucleotides using helicases.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe “strand sequencing” method, a single polynucleotide strand is passedthrough the pore and the identities of the nucleotides are derived.Strand sequencing can involve the use of a nucleotide handling protein,such as a helicase, to control the movement of the polynucleotidethrough the pore.

SUMMARY OF THE INVENTION

Spacers in polynucleotides are typically capable of stalling helicases,i.e. preventing helicases from moving further along the polynucleotidespast the spacers. The inventors have surprisingly demonstrated that itis possible to move one or more stalled helicases past a spacer in apolynucleotide by contacting the helicase and polynucleotide with atransmembrane pore and applying a potential. Since the helicase istypically too large to fit through the pore, the force of thepolynucleotide moving through the pore along the potential moves thehelicase past the spacer. This has important applications forcontrolling the movement of polynucleotides and characterising, such assequencing, polynucleotides. The inventors have also surprisinglydemonstrated that it is possible to control the loading of one or morehelicases on a polynucleotide using one or more spacers.

The invention therefore provides a method of moving one or more stalledhelicases past one or more spacers in a target polynucleotide,comprising contacting (a) the one or more stalled helicases and thetarget polynucleotide with a transmembrane pore and (b) applying apotential across the pore and thereby moving the one or more helicasespast the one or more spacers on the target polynucleotide.

The invention also provides:

-   -   a method of controlling the movement of a target polynucleotide        through a transmembrane pore, comprising (a) providing the        target polynucleotide with one or more spacers; (b) contacting        the target polynucleotide with one or more helicases such that        the one or more helicases stall at the one or more spacers; (c)        contacting the target polynucleotide and the one or more stalled        helicases with the pore; and (d) applying a potential across the        pore such that the one or more helicases move past the one or        more spacers and control the movement of the target        polynucleotide through the pore;    -   a method of characterising a target polynucleotide,        comprising (a) carrying out the method of controlling the        movement of a target polynucleotide through a transmembrane pore        of the invention; and (b) taking one or more measurements as the        polynucleotide moves with respect to the pore wherein the        measurements are indicative of one or more characteristics of        the polynucleotide and thereby characterising the target        polynucleotide;    -   a method of controlling the loading of one or more helicases on        a target polynucleotide, comprising (a) providing the        polynucleotide with one or more spacers; and (b) contacting the        polynucleotide provided in (a) with the one or more helicases        such that the one or more helicases bind to the polynucleotide        and stall at each spacer;    -   an adaptor for controlling the movement of a target        polynucleotide, wherein the adaptor comprises (a) (L-S-D)n or        (D-S-L)n in the 5′ to 3′ direction, wherein L is a single        stranded polynucleotide or a non-hybridised polynucleotide, S is        a spacer and D is a double stranded polynucleotide and wherein n        is a whole number and (b) one or more helicases stalled on each        adaptor; and    -   a kit for controlling the movement of a target polynucleotide,        wherein the kit comprises (a) one or more spacers, (b) one or        more helicases and (c) a transmembrane pore.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram of the lambda DNA construct used in Example 1.SEQ ID NO: 9 (labelled A) is attached at its 3′ end to four iSpC3spacers (labelled B). The four iSpC3 spacers are attached to the 5′ endof SEQ ID NO: 10 (labelled C). SEQ ID NO: 10 is attached to four iSpC3spacers (labelled D) which are attached to SEQ ID NO: 11 (labelled E) atits 5′ end. SEQ ID NO: 10 is hybridised to SEQ ID NO: 12 (labelled F,which has a 3′ cholesterol tether).

FIG. 2 shows an example current trace (y-axis label=Current (pA, 20 to120), x-axis label=Time (s, 3500 to 8000)) of when a helicase (T4Dda-E94C/A360C (SEQ ID NO: 8 with mutations E94C/A360C and then(ΔM1)G1G2))) controls the translocation of the Lambda DNA construct (0.2nM as described and illustrated in FIG. 1) through a nanopore(MS(B1-G75S/077S/L88N/Q126R)8 MspA (MspA-B2C) (SEQ ID NO: 2 withmutations 075S/G77S/L88N/Q126R)).

FIG. 3 shows zoomed in regions of the helicase-controlled DNA movementshown in the current trace in FIG. 2 (y-axis label=Current (pA, uppertrace 20 to 80, lower trace 20 to 60), x-axis label=Time (s, upper trace2995 to 3020, lower trace 8140 to 8170) for both the upper and lowertraces). A) shows the beginning of the helicase-controlled DNA movementand B) shows the end of the helicase controlled DNA movement. The arrowlabelled 1 corresponds to when the first four iSpC3 spacers (which isused to stall the movement of the enzyme prior to capture by thenanopore) moves through the nanopore. The arrow labelled 2 correspondsto a second group of four iSpC3 spacers moving through the nanopore.

FIG. 4 (a) shows the hairpin and Y-shaped MuA substrate designs used inExample 2. The dUMP in SEQ ID NO: 19 is highlighted as a triangle andthe iSpC3 spacers are shown as x's. FIG. 4 (b) shows the Lambda DNAconstruct produced during the sample preparation procedure detailed inExample 2. The 5-10 kB fragment of Lambda DNA is labelled X, thefragment of DNA filled in by the polymerase and joined to the rest ofthe construct by the ligase is labelled y (and is shown as a dottedline) and the iSpC3 spacers are shown as x's. A tether sequence (SEQ IDNO: 16) is hybridised to the DNA construct as shown. Attached t the 3′end of SEQ ID NO: 16 is six iSp18 spacers attached to two thymineresidues and a 3′ cholesterol TEG (shown as a grey circle).

FIG. 5 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both upper and lower traces) of when a helicase (TrwcCba (SEQ ID NO: 9) controls the translocation of the Lambda DNAconstruct (shown in FIG. 4b ) through a nanopore(MS(B1-G75S/G77S/L88N/Q126R)8 MspA (SEQ ID NO: 2 with mutationsG75S/G77S/L88N/Q126R)). The upper trace shows helicase controlled DNAmovement of the entire lambda DNA construct through the nanopore, thefirst iSpC3 spacer labelled X1 produces the spike in current labelled 1and the second iSpC3 spacers labelled X2 produces the spike in currentlabelled 2. The lower trace shows a zoomed in region of the end of thehelicase controlled DNA movement through the nanopore, the third iSpcC3spacer labelled X3 produces the spike in current labelled 3.

FIG. 6 (a) shows the hairpin and Y-shaped MuA substrates designs used inExample 3. The 5′ phosphate is labelled as a circle, the inosines in SEQID NO: 18 are highlighted as a rectangle and the iSpC3 spacers are shownas x's. FIG. 6 (b) shows the Lambda DNA construct produced during thesample preparation procedure detailed in Example 3. The 5-10 kB fragmentof Lambda DNA is labelled X, the inosines which have now been attachedto x are labelled as a rectangle and the iSpC3 spacers are shown as x's.A tether sequence (SEQ ID NO: 16) is hybridised to the DNA construct asshown. Attached t the 3′ end of SEQ ID NO: 16 is six iSp18 spacersattached to two thymine residues and a 3′ cholesterol TEG (shown as agrey circle).

FIG. 7 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both upper and lower traces) of when a helicase (TrwcCba (SEQ ID NO: 17) controls the translocation of the Lambda DNAconstruct (shown in FIG. 6b ) through a nanopore(MS(B1-G75S/G77S/L88N/Q126R)8 MspA (SEQ ID NO: 2 with mutationsG75S/G77S/L88N/Q126R)). The upper trace shows helicase controlled DNAmovement of the entire lambda DNA construct through the nanopore, thefirst iSpC3 spacer labelled X1 produces the spike in current labelled 1,the second iSpC3 spacer labelled X2 produces the spike in currentlabelled 2 and the third iSpC3 spacer labelled X3 produces the spike incurrent labelled 3. The lower trace shows a zoomed in region of thesecond half of the helicase controlled DNA movement through thenanopore, the second iSpC3 spacer labelled X2 produces the spike incurrent labelled 2 and the third iSpC3 spacer labelled X3 produces thespike in current labelled 3.

FIG. 8 Fluorescence assay for testing enzyme activity. A customfluorescent substrate was used to assay the ability of the helicase(labelled a) to displace hybridised dsDNA. 1) The fluorescent substratestrand (48.75 nM final, SEQ ID NO: 25 and 26) has a 3′ ssDNA overhang(20 bases), and a 40 base section of hybridised dsDNA. The upper strand(b) has a carboxyfluorescein base (c) at the 5′ end of SEQ ID NO: 25,and the hybridised complement (d) has a black-hole quencher (BHQ-1) base(e) at the 3′ end of SEQ ID NO: 26. When hybridised, the fluorescencefrom the fluorescein is quenched by the local BHQ-1, and the substrateis essentially non-fluorescent. 0.975 μM of a capture strand (f, SEQ IDNO: 27) that is part-complementary to the lower strand of thefluorescent substrate is included in the assay. 2) In the presence ofATP (0.975 mM) and MgCl₂ (10 mM), helicase Hel308 Mbu (12 nM, SEQ ID NO:28) binds to the 3′ overhang of the fluorescent substrate, moves alongthe upper strand, and displaces the complementary strand (d) as shown.3) Once the complementary strand with BHQ-1 is fully displaced thefluorescein on the major strand fluoresces (shown as a star shape). 4)The displaced lower strand (d) preferentially anneals to an excess ofcapture strand (f) to prevent re-annealing of initial substrate and lossof fluorescence.

FIG. 9 Fluorescence assay for testing enzyme activity. Two possiblefluorescent substrates were used to assay the ability of the helicase(labelled a) to displace hybridised dsDNA, these are shown in FIG. 9 (a)(labelled 1C-3C) has four Sp9 spacers (shown as a black triangle)connecting the 3′ end of SEQ ID NO: 27 to the 3′ end of SEQ ID NO: 29and (b) (labelled 1B-3B) has one Sp9 spacer (shown as a black triangle)connecting the 3′ end of SEQ ID NO: 27 to the 3′ end of SEQ ID NO: 29.The fluorescent substrate strand (48.75 nM final either a) or b)described previously) has a 3′ ssDNA overhang (20 bases), and a 40 basesection of hybridised dsDNA. The upper strand (b) has acarboxyfluorescein base (c) at the 5′ end of SEQ ID NO: 27, and thehybridised complement (d) has a black-hole quencher (BHQ-1) base (e) atthe 3′ end of SEQ ID NO: 26. When hybridised, the fluorescence from thefluorescein is quenched by the local BHQ-1, and the substrate isessentially non-fluorescent. 1 μM of a capture strand (f, SEQ ID NO: 27)that is part-complementary to the lower strand of the fluorescentsubstrate is included in the assay. 2 a and b) In the presence of ATP(0.975 mM) and MgCl₂ (10 mM), helicase Hel308 Mbu (SEQ ID NO: 28) bindsto the 3′ overhang of the fluorescent substrate, moves along the upperstrand up to the Sp9 group(s). The sp9 group (1 in B and 4 in C) stopsthe helicase from moving past it and the helicase does not displace thecomplementary strand (SEQ ID NO: 26). Therefore, the fluorescein andblack hole quencher remain in close proximity to each other andfluorescence is not observed.

FIG. 10 Graph (y-axis=Relative dsDNA turnover, x-axis=fluorescentsubstrate) of the relative dsDNA turnover rate in buffer solutions (100mM HEPES pH8, 0.975 mM ATP, 10 mM MgCl₂, 1 mg/mL BSA, 48.75 nMfluorescent substrate DNA (A=SEQ ID NOs: 25 and 26, B=SEQ ID NO: 27attached at its 3′ end by one Sp9 spacers to the 5′ end of SEQ ID NO: 29and hybridised to SEQ ID NO: 26, C=SEQ ID NO: 27 attached at its 3′ endby four Sp9 spacers to the 5′ end of SEQ ID NO: 29 and hybridised to SEQID NO: 26), 0.975 μM capture DNA (SEQ ID NO: 27)) for the Hel308 Mbuhelicase (labeled A, SEQ ID NO: 28) at 400 mM of KCl.

FIG. 11 Graph (y-axis=Relative dsDNA turnover, x-axis=fluorescentsubstrate) of the relative dsDNA turnover rate in buffer solutions (100mM HEPES pH8, 0.975 mM ATP, 10 mM MgCl₂, 1 mg/mL BSA, 48.75 nMfluorescent substrate DNA (D=SEQ ID NOs: 32 and 26, E=SEQ ID NO: 27attached at its 3′ end by one idSp groups to the 5′ end of SEQ ID NO: 30and hybridised to SEQ ID NO: 26, F=SEQ ID NO: 27 attached at its 3′ endby four idSp groups to the 5′ end of SEQ ID NO: 31 and hybridised to SEQID NO: 26 and G=SEQ ID NO: 33 hybridised to SEQ ID NO: 26), 0.975 μMcapture DNA (SEQ ID NO: 27)) for the Hel308 Mbu helicase (labeled A, SEQID NO: 28) at 400 mM of KCl.

FIG. 12 shows the experimental steps for the control strand which doesnot contain iSpC3 or iSp18 spacers which stall the helicase (labelled1). The control strand (SEQ ID NO: 34) contains no spacers or blockinggroups and is hybridised to a shorter complementary strand of DNA (SEQID NO: 35, which has a carboxyfluorescein attached to its 5′ end, shownas a grey circle). This produces a partially double stranded constructwhich has a 50 nucleotide overhang. The construct (SEQ ID NO: 34hybridised to SEQ ID NO: 35) is pro incubated with T4 Dda-E94C/A360C toallow the enzyme to bind to the overhang. Owing to the length of theoverhang more than one enzyme can bind to it as shown. The enzyme isthen provided with the necessary components to promote helicase movement(ATP and MgCl2). Additional capture strand (SEQ ID NO: 37) is also addedwith the ATP and MgCl2. The helicase then translocates along the controlstrand, displacing the shorter complementary strand, leaving the controlstrand with no helicase or complementary strand bound (labelled A). Thecomplementary strand then forms a hairpin so that it cannot re-anneal tothe control strand (labelled B). Helicase which is free in solution orhas moved along the control DNA and fallen off at the end is then boundby the excess of capture strand (SEQ ID NO: 37, complex labelled C) asshown. Any DNA which does not bind a helicase remains intact (remains asspecies F). The sample mixture is then run on a gel and the separatespecies identified by the bands they produce on the gel.

FIG. 13 shows the experimental steps for a strand which contains iSpC3or iSp18 spacers (shown as X's in the figure) in order to stall thehelicase (labelled 1). The same procedure was carried out as shown inFIG. 12, the enzyme was pre-incubated with the DNA construct (theshorter complementary strand has a carboxyfluorescein attached to its 5′end). The spacer groups are located in front of the double strandedregion and the helicase is capable of binding to the DNA as shown. Uponthe addition of ATP, MgCl2 and capture DNA (labelled 2) the helicase isthen provided with the necessary components to promote helicasemovement. If the spacers are capable of stalling the helicase then itwill remain bound to the DNA construct which still contains the shortcomplementary strand (labelled D). If the spacers are not capable ofstalling the helicase then it will move past the spacer and displace thecomplementary strand (labelled B) leaving species A with no helicasebound. The free enzyme will then bind to the excess capture strand(labelled C) and the displaced complementary strand will form a hairpin(labelled B). If the spacers are able to stall one helicase but not twohelicases then the first helicase will be pushed past the spacers by thehelicase behind and will displace the complementary stand. However, thesecond helicase will not be able to move past the spacers resulting inthe complex labelled E. Any DNA which does not bind a helicase remainsintact with no helicase bound (species F) The sample mixture is then runon a gel and the separate species identified by the bands they produceon the gel.

FIG. 14 shows the gel assay which was run for the control strand (1 intable 11). The lane labelled M shows a DNA ladder for reference (bandscorrespond from lowest mass (bottom of the gel) to highest mass (top ofthe gel) 200 bp (base pairs), 300 bp, 400 bp, 500/517 bp, 600 bp, 700bp, 800 bp, 900 bp, 1000 bp, 1200 bp and 1517 bp). Lane 1 containsannealed DNA only (SEQ ID NO: 34 hybridised to SEQ ID NO: 35). Lane 2contains the helicase (T4 Dda-E94C/A360C) pre-bound to control strand(no fuel added). Lane 3 shows the control strand after fuel (ATP andMgCl2) has been added in buffer 1. Lane 4 shows the control strand afterfuel (ATP and MgCl2) has been added in buffer 2. Band X corresponds toSEQ ID NO: 34 only and band Y corresponds to SEQ ID NO: 34 hybridised toSEQ ID NO: 35. The region labelled 1Y corresponds to one helicase boundto SEQ ID NO: 34 hybridised to SEQ ID NO: 35. The region labelled 2Ycorresponds to two helicases bound to SEQ ID NO: 34 hybridised to SEQ IDNO: 35. The region labelled 3Y corresponds to three helicases bound toSEQ ID NO: 34 hybridised to SEQ ID NO: 35. The region labelled 4Ycorresponds to four helicases bound to SEQ ID NO: 34 hybridised to SEQID NO: 35. The region labelled 5Y corresponds to five helicases bound toSEQ ID NO: 34 hybridised to SEQ ID NO: 35.

FIG. 15 shows the gel assay which was run for the DNA constructscontaining 3 (7 in table 11, corresponding lanes=1-4), 4 (8 in table 11,corresponding lanes=5-8) and 5 iSp18 spacers (9 in table 11,corresponding lanes=9-12) at the junction between ssDNA and dsDNA. Thelanes labelled M show a DNA ladder for reference (bands correspond fromlowest mass (bottom of the gel) to highest mass (top of the gel) 200 bp(base pairs), 300 bp, 400 bp, 500/517 bp, 600 bp, 700 bp, 800 bp, 900bp, 1000 bp, 1200 bp and 1517 bp). Lane 1 contains annealed DNA only(SEQ ID NO: 9 attached at its 3′ end to three iSp18 spacers which areattached to the 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35).Lane 2 contains the helicase (T4 Dda-E94C/A360C) pre-bound to the DNAconstruct with no fuel added (SEQ ID NO: 9 attached at its 3′ end tothree iSp18 spacers which are attached to the 5′ end of SEQ ID NO: 36hybridised to SEQ ID NO: 35). Lane 3 shows the DNA construct (SEQ ID NO:9 attached at its 3′ end to three iSp18 spacers which are attached tothe 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35) after fuel (ATPand MgCl2) has been added in buffer 1. Lane 4 shows the DNA construct(SEQ ID NO: 9 attached at its 3′ end to three iSp18 spacers which areattached to the 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35)after fuel (ATP and MgCl2) has been added in buffer 2. Lane 5 containsannealed DNA only (SEQ ID NO: 9 attached at its 3′ end to four iSp18spacers which are attached to the 5′ end of SEQ ID NO: 36 hybridised toSEQ ID NO: 35). Lane 6 contains the helicase (T4 Dda-E94C/A360C)pre-bound to the DNA construct with no fuel added (SEQ ID NO: 9 attachedat its 3′ end to four iSp18 spacers which are attached to the 5′ end ofSEQ ID NO: 36 hybridised to SEQ ID NO: 35). Lane 7 shows the DNAconstruct (SEQ ID NO: 9 attached at its 3′ end to four iSp18 spacerswhich are attached to the 5′ end of SEQ ID NO: 36 hybridised to SEQ IDNO: 35) after fuel (ATP and MgCl2) has been added in buffer 1. Lane 8shows the DNA construct (SEQ ID NO: 9 attached at its 3′ end to fouriSp18 spacers which are attached to the 5′ end of SEQ ID NO: 36hybridised to SEQ ID NO: 35) after fuel (ATP and MgCl2) has been addedin buffer 2. Lane 9 contains annealed DNA only (SEQ ID NO: 9 attached atits 3′ end to five iSp18 spacers which are attached to the 5′ end of SEQID NO: 36 hybridised to SEQ ID NO: 35). Lane 10 contains the helicase(T4 Dda-E94C/A360C) pre-bound to the DNA construct with no fuel added(SEQ ID NO: 9 attached at its 3′ end to five iSp18 spacers which areattached to the 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35).Lane 11 shows the DNA construct (SEQ ID NO: 9 attached at its 3′ end tofive iSp18 spacers which are attached to the 5′ end of SEQ ID NO: 36hybridised to SEQ ID NO: 35) after fuel (ATP and MgCl2) has been addedin buffer 1. Lane 12 shows the DNA construct (SEQ ID NO: 9 attached atits 3′ end to five iSp18 spacers which are attached to the 5′ end of SEQID NO: 36 hybridised to SEQ ID NO: 35) after fuel (ATP and MgCl2) hasbeen added in buffer 2. Band X corresponds to ssDNA construct only (e.g.SEQ ID NO: 9 attached at its 3′ end to three four or five iSp18 spacerswhich are attached to the 5′ end of SEQ ID NO: 36) only and Band Ycorresponds to the dsDNA construct only (SEQ ID NO: 9 attached at its 3′end to three, four or five iSp18 spacers which are attached to the 5′end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35). The region labelled1X corresponds to one helicase bound to SEQ ID NO: 9 attached at its 3′end to three, four or five iSp18 spacers which are attached to SEQ IDNO: 36. The region labelled 1Y corresponds to one helicase bound to SEQID NO: 9 attached at its 3′ end to three, four or five iSp18 spacerswhich are attached to SEQ ID NO: 36 hybridised to SEQ ID NO: 35. Theregion labelled 2Y corresponds to two helicases bound to SEQ ID NO: 9attached at its 3′ end to three, four or five iSp18 spacers which areattached to SEQ ID NO: 36 hybridised to SEQ ID NO: 35. The regionlabelled 3Y corresponds to three helicases bound to SEQ ID NO: 9attached at its 3′ end to three, four or five iSp18 spacers which areattached to SEQ ID) NO: 36 hybridised to SEQ ID NO: 35. The regionlabelled 4Y corresponds to four helicases bound to SEQ ID NO: 9 attachedat its 3′ end to three, four or five iSp18 spacers which are attached toSEQ ID NO: 36 hybridised to SEQ ID NO: 35. The region labelled 5Ycorresponds to five helicases bound to SEQ ID NO: 9 attached at its 3′end to three, four or five iSp18 spacers which are attached to SEQ IDNO: 36 hybridised to SEQ ID NO: 35.

FIG. 16 shows the standard DNA construct used in Example 9. The x'srepresent spacer groups onto which the helicase cannot bind. The lengthof the region labelled 1 can be altered in order to control the numberof helicases which can bind to that region. The figure shows, as anexample, the binding of one or two helicases in region 1.

FIG. 17 shows an example gel assay of the DNA construct labelled 3 inTable 12 (Five iSpC3 spacers attached to the 5′ end of SEQ ID NO: 9,which is attached at its 3′ end to four iSpC3 spacers which are attachedto the 5′ end of SEQ ID NO: 36 which is hybridised to SEQ ID NO: 35).The lane labelled M shows a DNA ladder for reference (bands correspondfrom lowest mass (bottom of the gel) to highest mass (top of the gel)200 bp (base pairs), 300 bp, 400 bp, 500/517 bp, 600 bp, 700 bp, 800 bp,900 bp, 1000 bp, 1200 bp and 1517 bp). Lanes 1-6 correspond to differentconcentrations of T4 Dda-E94C/A360C-1=5000 nM, 2=2500 nM, 3=1250 nM,4=625 nM, 5=312.5 nM and 6=0 nM). The band observed at level Xcorresponds to the unbound dsDNA construct. The numbers on the left-handside of the gel relate to the number of enzymes bound to the DNA. Forthis DNA construct it was possible to bind up to 6 helicases at thehighest concentration of enzyme added. The numbers shown at the top ofthe gel correspond to the concentration of T4 Dda-E94C/A360C) added.

FIG. 18 shows the DNA construct used in example 9. Regions labelled Aand B are short strands of DNA (SEQ ID NO: 37) which have been designedso that one helicase can bind to each region (as shown on the right-handside). Region 1 corresponds to 25 SpC3 spacers, region 2 corresponds totwo iSp18 spacers, region 3 corresponds to two iSp18 spacers, region 4is a section of DNA (SEQ ID NO: 10) which hybridises to another strandof DNA which may or may not be made up of forked DNA (e.g. SEQ ID NO:42=non-forked (missing fragment labelled 7) and SEQ ID NO: 12 attachedto six iSp18 spacers (shown as fragment labelled 7)=forked), region 5corresponds to four 5-nitroindoles and region 6 corresponds to anotherregion of DNA (SEQ ID NO: 41) which is hybridised to its complement (SEQID NO: 43).

FIG. 19 shows a gel assay of the DNA construct described and shown inFIG. 18. The band observed at level X corresponds to the unbound dsDNAconstruct. The lane labelled M shows a DNA ladder for reference (bandscorrespond from lowest mass (bottom of the gel) to highest mass (top ofthe gel) 200 bp (base pairs), 300 bp, 400 bp, 500/517 bp, 600 bp, 700bp, 800 bp, 900 bp, 1000 bp, 1200 bp and 1517 bp). The numbers on theleft-hand side of the gel relate to the number of enzymes bound to theDNA. For this DNA construct it was possible to observe binding of twohelicases (one at region A and the second at region B as shown in FIG.18) from a concentration of 475 nM enzyme and above. The numbers shownat the top of the gel correspond to the concentration of T4Dda-E94C/A360C) added.

FIG. 20 shows the DNA construct used in Example 10 (referred to as DNAconstruct X1). There are 25 SpC3 spacers (shown as x's) attached to the5′ end of SEQ ID NO: 38 which is attached at its 3′ end to 4 iSp18spacers (shown as black squares). The four iSp18 spacers are attached tothe 5′ end of SEQ ID NO: 10 which is attached at its 3′ end to four5-nitroindoles (shown as grey triangles). The four nitroindoles are thenattached to the 5′ end of SEQ ID NO: 41. The complementary DNA strandwhich hybridises to SEQ ID NO: 41 is SEQ ID NO: 43. The complementaryDNA strand which hybridises to SEQ ID NO: 10 is SEQ ID NO: 12 (attachedto SEQ ID NO: 12's 3′ end is six iSp18 spacers attached to two thymineresidues and a 3′ cholesterol TEG (shown as a grey circle)). The regionlabelled A corresponds to the region of the construct where T4Dda-E94C/A360C is able to bind.

FIG. 21 shows an example current trace (y-axis label=Current (pA, 50 to250), x-axis label=Time (s, 238 to 252)) of when a helicase (T4Dda-E94C/A360C) controls the translocation of the DNA construct (0.1 nM,see FIG. 20 description) through a nanopore (MspA-B2C). The regionlabelled 1 shows the helicase controlled translocation of the polyTregion (SEQ ID NO: 38, that the enzyme bound onto) through the nanopore.The region labelled 2 corresponds to the helicase controlledtranslocation of the iSp18 spacers through the nanopore.

FIG. 22 shows a diagram of the DNA construct used in Example 4. Twothymines are attached at the 3′ end to 28 iSpC3 spacers (labelled A).The 28 iSpC3 spacers are attached at the other end to the 5′ end of SEQID NO: 23 (sequence corresponds to region B=polyT section and regionC=sequence complementary to the tether sequence (SEQ ID NO: 12)). The 3′end of SEQ ID NO: 23 is attached to four iSpC3 spacers (labelled D). Theother end of the 4 iSpC3 spacers is attached to the 5′ end of SEQ ID NO:24. The tether sequence (SEQ ID NO: 12) is attached at its 3′ end to sixiSp18 spacers attached to two thymine residues and a 3′ cholesterol TEG.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe MS-B1 mutant MspA monomer. This mutant lacks the signal sequence andincludes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of theMS-B1 mutant of the MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the amino acid sequence of the helicase Dda 1993 fromEnterobactcria phage T4.

SEQ ID NO: 9 shows a polynucleotide sequence used in Example 1, 2, 3, 7and 8.

SEQ ID NO: 10 shows a polynucleotide sequence used in Example 1 and 9.

SEQ ID NO: 11 shows a polynucleotide sequence used in Example 1. SEQ IDNO: 11 is attached by its 5′ end to three iSpC3 spacers which areattached to the 3′ end of SEQ ID NO: 10.

SEQ ID NO: 12 shows a polynucleotide sequence used in Examples 1 and 9.In Example 1 SEQ ID NO: 12 is attached at its 3′ end to six iSp18spacers attached to two thymine residues and a 3′ cholesterol TEG. InExample 9 SEQ ID NO: 12 is attached at its 3′ end to six iSp18 spacersonly.

SEQ ID NO: 13 shows the polynucleotide sequence of the Entcrobacteriaphage λ. The sequence contains an additional 12 base overhang attachedat the 5′ end of the sense strand. The sequence shown here is that ofthe sense strand only.

SEQ ID NO: 14 shows a polynucleotide sequence used in Examples 2 and 3.SEQ ID NO: 14 is attached at its 3′ end to the 5′ end of SEQ ID NO: 15by four iSpC3 spacer units.

SEQ ID NO: 15 shows a polynucleotide sequence used in Examples 2 and 3.SEQ ID NO: 15 is attached at its 5′ end to the 3′ end of SEQ ID NO: 14by four iSpC3 spacer units.

SEQ ID NO: 16 shows a polynucleotide sequence used in Example 2 and 3which at the 3′ end of the sequence has six iSp18 spacers attached totwo thymine residues and a 3′ cholesterol TEG.

SEQ ID NO: 17 shows the amino acid sequence of the Trwc Cba helicase.

SEQ ID NO: 18 shows a polynucleotide sequence used in Example 3. SEQ IDNO: 18 is attached at its 3′ end to the 5′ end of SEQ ID NO: 9 by fouriSpC3 spacer units. This sequence has a phosphate attached to its 5′ endand 5 deoxyinosines at positions 1 to 5.

SEQ ID NO: 19 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 20 shows a polynucleotide sequence used in Example 2.

SEQ ID NOs: 21 and 22 are placeholders to maintain the numbering of thefollowing sequences.

SEQ ID NO: 23 shows the polynucleotide sequence used in Example 4.Attached to the 5′ end of this sequence is 28 iSpC3 spacers units thelast of which has an additional two T's attached to the 5′ end of thespacer group. Attached to the 3′ end of this sequence is four iSpC3spacer units which are attached to the 5′ end of SEQ ID NO: 24.

SEQ ID NO: 24 shows the polynucleotide sequence used in Example 9.Attached to the 5′ end of this sequence is four iSpC3 spacer units, thelast of which is attached to SEQ ID NO: 23. Attached to the 5′ end ofSEQ ID NO: 23 is 28 iSpC3 spacer units the last of which has anadditional two T's attached to the 5′ end of the spacer group.

SEQ ID NO: 25 shows a polynuclcotide sequence used in Example 5. It hasa carboxyfluorescein (FAM) base at its 5′ end.

SEQ ID NO: 26 shows a polynucleotide sequence used in Example 5 and 6.It has a black-hole quencher (BHQ-1) base at its 3′ end.

SEQ ID NO: 27 shows a polynucleotide sequence used in Examples 5 and 6.

SEQ ID NO: 28 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 29 shows a polynucleotide sequence used in Example 5. Thissequence is connected to SEQ ID NO: 27 at its 5′ end by either one orfour iSp9 spacer groups.

SEQ ID NO: 30 shows a polynucleotide sequence used in Example 6. Thissequence is connected to SEQ ID NO: 27 at its 5′ end by one idSp group.

SEQ ID NO: 31 shows a polynucleotide sequence used in Example 6. Thissequence is connected to SEQ ID NO: 27 at its 5′ end by four idSpgroups.

SEQ ID NO: 32 shows a polynucleotide sequence used in Example 6. It hasa carboxyfluorescein (FAM) base at its 5′ end.

SEQ ID NO: 33 shows a polynucleotide sequence used in Example 6. It hasa carboxyfluorescein (FAM) base at its 5′ end.

SEQ ID NO: 34 shows a polynucleotide sequence used in Example 7.

SEQ ID NO: 35 shows a polynucleotide sequence used in Example 7 and 8.It has a carboxyfluorescein (FAM) base at its 5′ end.

SEQ ID NO: 36 shows a polynucleotide sequence used in Example 7 and 8.

SEQ ID NO: 37 shows a polynucleotide sequence used in Example 7, 8 and9.

SEQ ID NO: 38 shows a polynucleotide sequence used in Example 8 and 10.

SEQ ID NO: 39 shows a polynucleotide sequence used in Example 8.

SEQ ID NO: 40 shows a polynucleotide sequence used in Example 8.

SEQ ID NO: 41 shows a polynucleotide sequence used in Example 9.

SEQ ID NO: 42 shows a polynuclcotide sequence used in Example 9.

SEQ ID NO: 43 shows a polynucleotide sequence used in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynuclcotides, reference to “aspacer” includes two or more spacers, reference to “a helicase” includestwo or more helicases, reference to “a transmembrane pore” includes twoor more pores, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Methods of Moving Helicases Past Spacers

The invention provides a method of moving one or more stalled helicasespast one or more spacers in a target polynucleotide. A helicase isstalled if it has stopped moving along the polynucleotide. Each spacertypically stalls the one or more helicases. Methods for determiningwhether or not one or more helicases are stalled are discussed below.The one or more helicases may be stalled before a spacer. The one ormore helicases may be stalled by a spacer. The one or more helicases maybe stalled on a spacer. The invention concerns moving the one or morestalled helicases past, i.e. beyond, the one or more spacers.

The one or more stalled helicases and the target polynucleotide arecontacted with a transmembrane pore and a potential is applied. Asdescribed in more detail below, the target polynucleotide moves throughthe pore with the field resulting from the applied potential. The one ormore helicases are typically too large to move through the pore. When apart of the target polynucleotide enters the pore and moves through thepore along the field resulting from the applied potential, the one ormore helicases are moved past the spacer by the pore as the targetpolynucleotide moves through the pore. This is because the targetpolynucleotide (including the one or more spacers) moves through thepore and the one or more helicases remain on top of the pore.

This allows the position of the one or more helicases on the targetpolynucleotide to be controlled. Before the one or more stalledhelicases and the target polynucleotide are contacted with atransmembrane pore and the potential is applied, the one or morehelicases remain in the position where they are stalled. Even in thepresence of the necessary components to facilitate helicase movement(e.g. ATP and Mg²⁺), the one or more helicases will not move past aspacer on the target polynucleotide and will not move along the portionof the target polynucleotide on other side of the spacer until they arein the presence of the transmembrane pore and the applied potential.

The one or more helicases will also remain in the position where theyare stalled in the presence of the transmembrane pore, but in theabsence of the applied potential. In this instance, the application of apotential moves the one or more helicases past a spacer. The applicationof a potential can therefore be used to instigate the movement of thethe one or more helicases past a spacer and along the portion of thetarget polynucleotide on other side of the spacer. For instance, anincrease in voltage may be used to move the one or more helicases pastthe spacer.

The invention also provides a method of controlling the movement of atarget polynucleotide through a transmembrane pore. The targetpolynucleotide is provided with one or more spacers. The targetpolynucleotide is contacted with one or more helicases and the one ormore helicases stall at the one or more spacers. This ensures that theone or more helicases remain at one or more specific positions on thepolynucleotide. This is discussed in more detail below. The targetpolynucleotide and the one or more stalled helicases are contacted witha transmembrane pore. Once a potential is applied, the one or morehelicases move past the one or more spacers and along the portion of thepolynucleotide on other side of the spacer(s). This allows the one ormore helicases to control the movement of the polynucleotide through thepore. The potential is also typically used to thread the polunucleotideinto the pore.

Helicases can control the movement of polynucleotides in at least twoactive modes of operation (when the helicase is provided with all thenecessary components to facilitate movement, e.g. ATP and Mg²⁺) and oneinactive mode of operation (when the helicase is not provided with thenecessary components to facilitate movement). When provided with all thenecessary components to facilitate movement, the helicase moves alongthe polynucleotide in a 5′ to 3′ or a 3′ to 5′ direction (depending onthe helicase), but the orientation of the polynucleotide in the pore(which is dependent on which end of the polynucleotide is captured bythe pore) means that the helicase can be used to either move thepolynucleotide out of the pore against the applied field or move thepolynucleotide into the pore with the applied field. When the end of thepolynucleotide towards which the helicase moves is captured by the pore,the helicase works against the direction of the field resulting from theapplied potential and pulls the threaded polynucleotide out of the poreand into the cis chamber. However, when the end away from which thehelicase moves is captured in the pore, the helicase works with thedirection of the field resulting from the applied potential and pushesthe threaded polynucleotide into the pore and into the trans chamber.

When the helicase is not provided with the necessary components tofacilitate movement it can bind to the polynucleotide and act as a brakeslowing the movement of the polynucleotide when it is pulled into thepore by the field resulting from the applied potential. In the inactivemode, it does not matter which end of the polynucleotide is captured, itis the applied field which pulls the polynucleotide into the poretowards the trans side with the helicase acting as a brake. When in theinactive mode, the movement control of the polynucleotide by thehelicase can be described in a number of ways including ratcheting,sliding and braking.

In the method of the invention, the one or more helicases preferablycontrol the movement of the target polynucleotide through the pore withthe field resulting from the applied potential. In one preferredembodiment, the one or more helicases are used in the active mode andthe end away from which the one or more helicases move is captured bythe pore such that the one or more helicases work with the fieldresulting from the applied potential and push the polynucleotide throughthe pore. If the one or more helicases move in the 5′ to 3′ direction,the 5′ end of the target polynucleotide is preferably captured by thepore. In such embodiments, the one or more helicases are moved past theone or more spacers in the 5′ to 3′ direction. If the one or morehelicases move in the 3′ to 5′ direction, the 3′ end of the targetpolynucleotide is preferably captured by the pore. In such embodiments,the one or more helicases are moved past the one or more spacers in the3′ to 5′ direction.

In another preferred embodiment, the one or more helicases are used inthe inactive mode such that the applied field pulls the targetpolynucleotide through the pore and the one or more helicases act as abrake. In the method of the invention, the one or more helicasespreferably slow or brake the movement of the target polynucleotidethrough the pore with the field resulting from the applied potential. Ineither case, the one or more helicases are typically too large to movethrough the pore and the pore pushes the one or more helicases past theone or more spacers on the polynucleotide as the polynucleotide movesthrough the pore with the field resulting from the applied potential.

The method of controlling the movement of a target polynucleotidethrough a transmembrane pore can be helpful during characterisation ofthe polynucleotide using the pore, for instance during strandsequencing. The invention also provides a method of characterising atarget polynucleotide. The target polynucleotide is provided with one ormore spacers. The target polynucleotide is contacted with one or morehelicases and the one or more helicases stall at the one or morespacers. This ensures that the one or more helicases remain at one ormore specific positions on the polynucleotide. This is discussed in moredetail below. The target polynucleotide and the one or more stalledhelicases are contacted with a transmembrane pore. Once a potential isapplied, the one or more helicases move past the one or more spacers andalong the portion of the polynucleotide on other side of the spacer(s).This allows the one or more helicases to control the movement of thepolynucleotide through the pore. The method also comprises taking one ormore measurements as the polynucleotide moves with respect to the pore.The measurements are indicative of one or more characteristics of thepolynucleotide.

The ability to stall one or more helicases on the target polynucleotideand move the one or more helicases past the one or more spacers using atransmembrane pore and an applied potential is advantageous because itallows effective chareterisation, such as sequencing, of the targetpolynucleotide. For instance, the one or more helicases can be stalledtowards one end of the target polynucleotide in a leader sequence whichis designed to be captured by the pore and does not need to becharacterised (as described below). The stalling of the one or morehelicases in the leader sequence means that the one or more helicases donot move away from the leader sequence along the part of thepolynucleotide to be characterised until it/they are contacted with thepore and a potential is applied. Once the one or more helicases and thepolynucleotide are contacted with the pore and a potential is applied,the leader sequence is typically captured by the pore and moves throughthe pore. This movement moves the one or more helicases past thespacer(s) and along the part of polynucleotide to be characterised (asdescribed above). The one or more helicases may then control themovement of the part of the polynucleotide to be characterised.

If the one or more helicases are not stalled in the leader sequence,it/they would move along the polynucleotide away from the leadersequence and along the part of the polynucleotide to be characterised.When the one or more helicases and the polynucleotide are contacted withthe pore and a potential is applied under these circumstances, theleader sequence and some, if not all, of the polynucleotide to becharacterised will move in an uncontrolled manner through the pore alongthe field resulting from the applied potential. Only once the one ormore helicases come into contact with the pore will it/they begin tocontrol the movement of the part of the polynucleotide to becharacterised as discussed above. Any part of the polynucleotide whichmoves through the pore in an uncontrolled manner cannot be characterisedas described below. If the one or more helicases move away from theleader sequence and along most of the rest of the target polynucleotidelittle, if any, of the polynucleotide will be characterised.

The use of one or more spacers in accordance with the invention alsoallows the number and position of the one or more helicases on thetarget polynucleotide to be controlled as discussed in more detailbelow. For instance, a specific number of helicases may be stalled atspecific positions on adaptors which may be ligated to the targetpolynucleotide before characterisation. Such adaptors are provided bythe invention and may be provided in a kit for characterisation. The useof one or more spacers ensures that the helicases remain where theysupposed to be until the characterisation is begun, even if the adaptorand/or target polynucleotide before characterisation are in the presenceof the components necessary to facilitate helicase movement (e.g. ATPand Mg²⁺).

Polynucleotide

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the polynucleotidecan be oxidized or methylated. One or more nucleotides in thepolynucleotide may be damaged. For instance, the polynucleotide maycomprise a pyrimidine dimer. Such dimers are typically associated withdamage by ultraviolet light and are the primary cause of skin melanomas.One or more nucleotides in the polynucleotide may be modified, forinstance with a label or a tag. Suitable labels are described below.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase and sugar form a nucleoside.

The nucleobese is typically heterocyclic. Nucleobases include, but arenot limited to, purines and pyrimidines and more specifically adenine(A), guanine (0), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, butare not limited to, ribose and deoxyribose. The sugar is preferably adeoxyribose.

The nucleotide in the polynucleotide is typically a ribonucleotide ordeoxyribonucleotide. The polynucleotide may comprise the followingnucleosides: adenosine, uridine, guanosine and cytidine. The nuclcotideis preferably a deoxyribonucleotide. The polynucleotide preferablycomprises the following nucleosides: deoxyadenosine (dA), deoxyuridine(dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide typically contains a monophosphate, diphosphate ortriphosphate. Phosphates may be attached on the 5′ or 3′ side of anucleotide.

Suitable nucleotides include, but are not limited to, adenosinemonophosphate (AMP), guanosine monophosphate (GMP), thymidinemonophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate(CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyguanosine monophosphate (dUMP), deoxythymidine monophosphate(dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidinemonophosphate (dCMP). The nucleotides are preferably selected from AMP,TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP. The nucleotides aremost preferably selected from dAMP, dTMP, dGMP, dCMP and dUMP. Thepolynucleotide preferably comprises the following nucleotides: dAMP,dUMP and/or dTMP, dGMP and dCMP.

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide can be a nucleic acid. The polynucleotide may be anysynthetic nucleic acid known in the art, such as peptide nucleic acid(PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), lockednucleic acid (LNA) or other synthetic polymers with nucleotide sidechains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The polynucleotide preferably does not comprise any abasic nucleotides(i.e. nucleotides which lack a nucleobase), except in the one or morespacers. The polynucleotide preferably does not comprise any C3 spacers(i.e. nucleotide which lack a nucleobase and a sugar), except in the oneor more spacers.

The polynucleotide may be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 400 or at least 500nucleotides in length. The polynucleotide can be 1000 or morenucleotides, 5000 or more nucleotides in length or 100000 or morenucleotides in length.

The helicase may move along the whole or only part of the targetpolynucleotide in the method of the invention. The whole or only part ofthe target polynucleotide may be characterised using the method of theinvention.

The target polynucleotide may be single stranded. At least a portion ofthe target polynucleotide is preferably double stranded. Helicasestypically bind to single stranded polynucleotides. If at least a portionof the target polynucleotide is double stranded, the targetpolynucleotide preferably comprises a single stranded region or anon-hybridised region. The one or more helicases are capable of bindingto the single stranded region or one strand of the non-hybridisedregion. The target polynucleotide preferably comprises one or moresingle stranded regions or one or more non-hybridised regions.

The one or more spacers are preferably included in the single strandedregion or the non-hybridised region of the target polynucleotide. Thetarget polynucleotide may comprise more than one single stranded regionor more than one non-hybridised region. The target polynucleotide maycomprise a single stranded region or a non-hybridised region within itssequence and/or at one or both ends. The one or more spacers may beincluded in the double stranded region of the target polynucleotide.

If the one or more helicases used in the method move in the 5′ to 3′direction, the target polynucleotide preferably comprises a singlestranded region or a non-hybridised region at its 5′ end. If the one ormore helicases used in the method move in the 3′ to 5′ direction, thetarget polynucleotide preferably comprises a single stranded region or anon-hybridised region at its 3′ end. If the one or more helicases areused in the inactive mode (i.e. as a brake), it does not matter wherethe single stranded region or the non-hybridised region is located.

The single stranded region preferably comprises a leader sequence whichpreferentially threads into the pore. The leader sequence facilitatesthe method of the invention. The leader sequence is designed topreferentially thread into the transmembrane pore and thereby facilitatethe movement of target polynucleotide through the pore. The leadersequence typically comprises a polymer. The polymer is preferablynegatively charged. The polymer is preferably a polynucleotide, such asDNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA,polyethylene glycol (PEG) or a polypeptide. The leader preferablycomprises a polynucleotide and more preferably comprises a singlestranded polynucleotide. The leader sequence can comprise any of thepolynucleotides discussed above. The single stranded leader sequencemost preferably comprises a single strand of DNA, such as a poly dTsection. The leader sequence preferably comprises the one or morespacers.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 150 nucleotides in length. Thelength of the leader typically depends on the transmembrane pore used inthe method.

If at least a portion of the target polynucleotide is double stranded,the two strands of the double stranded portion are preferably linkedusing a bridging moiety, such as a hairpin. This facilitatescharacterisation method of the invention. Linking the two strands of thetarget polynuclcotide by a bridging moiety allows both strands of thepolynucleotide to be characterised, such as sequenced, by thetransmembrane pore. The two strands dchybridise as the polynucleotidemoves though the pore as a single stranded polynucleotide. This methodis advantageous because it doubles the amount of information obtainedfrom a single double stranded target polynucleotide. Moreover, becausethe sequence in the complementary ‘anti-sense’ strand is necessarilyorthogonal to the sequence of the ‘sense’ strand, the information fromthe two strands can be combined informatically. Thus, this mechanismprovides an orthogonal proof-reading capability that provides higherconfidence observations.

Any of the embodiments disclosed in International Application No.PCT/GB2012/051786 (published as WO 2013/014451) may be used. Thebridging moiety typically covalently links the two strands of thepolynucleotide. The bridging moiety can be anything that is capable oflinking the two strands of the target polynucleotide, provided that thebridging moiety does not interfere with movement of the polynucleotidethrough the transmembrane pore. Suitable bridging moieties include, butare not limited to a polymeric linker, a chemical linker, apolynucleotide or a polypeptide. Preferably, the bridging moietycomprises DNA, RNA, modified DNA (such as abasic DNA), RNA, PNA, LNA orpolyctheylone glycol (PEG). The bridging moiety is more preferably DNAor RNA. The bridging moiety may comprise the one or more spacers.

The bridging moiety is most preferably a hairpin loop. The hairpin loopmay be formed from any of the polynucleotides disclosed above. Thehairpin loop or the loop of the hairpin loop is typically from about 4to about 100 nucleotides in length, preferably from about 4 to about 8nucleotides in length.

The bridging moiety is linked to the two strands of the targetpolynucleotide by any suitable means known in the art. The bridgingmoiety may be synthesized separately and chemically attached orenzymatically ligated to the target polynucleotide. Alternatively, thebridging moiety may be generated in the processing of the targetpolynucleotide.

The bridging moiety is linked to the target polynucleotide at or nearone end of the target polynucleotide. The bridging moiety is preferablylinked to the target polynucleotide within 10 nucleotides of the end ofthe polynucleotide.

The one or more spacers are preferably positioned such that it/theystall(s) the one or more helicases and prevents it/them from movingalong the target polynucleotide to be controlled or characterised. Forinstance, the one or more spacers are preferably located between aleader sequence and the target polynucleotide to be controlled orcharacterised, for instance within a leader sequence at one end of thepolynucleotide. The leaders sequence typically enters the pore with thefield resulting from the applied potential and the one or more helicasesare moved past the one or more spacers as the polynucleotide movesthrough the pore. The one or more helicases may then control themovement of the remainder of the target polynucleotide through the poreand facilitate its characterisation.

In the most preferred embodiment, the target polynucleotide comprises adouble stranded portion which is linked at one end by a bridging moiety,such as a hairpin loop, and a single stranded portion at the at theother end from the bridging moiety which comprises a leader sequence.The one or more spacers may be present in the leader sequence and/or thebridging moiety.

The target polynucleotide is present in any suitable sample. Theinvention is typically carried out on a sample that is known to containor suspected to contain the target polynucleotide. The invention may becarried out on a sample to confirm the identity of one or more targetpolynucleotides whose presence in the sample is known or expected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaeal,prokaryotic or eukaryotic and typically belongs to one of the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep or pigs or may alternatively bepets such as cats or dogs. Alternatively a sample of plant origin istypically obtained from a commercial crop, such as a cereal, legume,fruit or vegetable, for example wheat, quinoa, barley, oats, canola,maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco,beans, lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example bycentrifugation or by passage through a membrane that filters outunwanted molecules or cells, such as red blood cells. The sample may bemeasured immediately upon being taken. The sample may also be typicallystored prior to assay, preferably below −70° C.

Spacer(s)

The one or more spacers are included in the target polynucleotide. Theone or more spacers are preferably part of the target polynucleotide,for instance it/they interrupt(s) the polynucleotide sequence. The oneor more spacers are preferably not part of one or more blockingmolecules, such as speed bumps, hybridised to the target polynucleotide.

There may be any number of spacers in the target polynucleotide, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more spacers. There are preferably two,four or six spacers in the target polynucleotide. There may be spacer indifferent regions of the target polynucleotide, such as a spacer in theleader sequence and a spacer in the hairpin loop.

The one or more spacers each provides an energy barrier which the one ormore helicases cannot overcome even in the active mode. The one or morespacers may stall the one or more more helicases by by reducing thetraction of the helicase (for instance by removing the bases from thenucleotides in the target polynucleotide) or physically blockingmovement of the one or more helicases (for instance using a bulkychemical group).

The one or more spacers may comprise any molecule or combination ofmolecules that stalls the one or more helicases. The one or more spacersmay comprise any molecule or combination of molecules that prevents theone or more helicases from moving along the target polynucleotide. It isstraightforward to determine whether or not the one or more helicasesare stalled at one or more spacers in the absence of a transmembranepore and an applied potential. For instance, this can be assayed asshown in the Examples, for instance the ability of a helicase to mvepast a spacer and displace a complementary strand of DNA can be measuredby PAGE.

The one or more spacers typically comprise a linear molecule, such as apolymer. The one or more spacers typically have a different structurefrom the target polynucleotide. For instance, if the targetpolynucleotide is DNA, the one or more spacers are typically not DNA. Inparticular, if the target polynucleotide is deoxyribonucleic acid (DNA)or ribonucleic acid (RNA), the one or more spacers preferably comprisepeptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleicacid (TNA), locked nucleic acid (LNA) or a synthetic polymer withnucleotide side chains.

The one or more spacers preferably comprises one or more nitroindoles,such as one or more 5-nitroindoles, one or more inosines, one or moreacridines, one or more 2-aminopurines, one or more 2-6-diaminopurines,one or more 5-bromo-deoxyuridines, one or more inverted thymidines(inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one ormore dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one ormore 5-hydroxymethylcytidines, one or more 2′-O-Methyl RNA bases, one ormore Iso-deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines(Iso-dGs), one or more iSpC3 groups (i.e. nucleotides which lack sugarand a base), one or more photo-cleavable (PC) groups, one or morehexandiol groups, one or more spacer 9 (iSp9) groups, one or more spacer18 (iSp18) groups, a polymer or one or more thiol connections. The oneor more spacers may comprise any combination of these groups. Many ofthese groups are commercially available from IDT® (Integrated DNATechnologies®).

The one or more spacers may contain any number of these groups. Forinstance, for 2-aminopurines, 2-6-diaminopurines, 5-bromo-deoxyuridines,inverted dTs, ddTs, ddCs, 5-methylcytidines, 5-hydroxymethylcytidines,2′-O-Methyl RNA bases, Iso-dCs, Iso-dGs, iSpC3 groups, PC groups,hexandiol groups and thiol connections, the one or more spacerspreferably comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The oneor more spacers preferably comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9groups. The one or more spacers preferably comprise 2, 3, 4, 5 or 6 ormore iSp18 groups. The most preferred spacer is four iSp18 groups.

The polymer is preferably a polypeptide or a polyethylene glycol (PEG).The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or more monomer units.

The one or more spacers preferably comprise one or more abasicnucleotides (i.e. nucleotides lacking a nucleobase), such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The nucleobase can bereplaced by —H (idSp) or —OH in the abasic nucleotide. Abasic spacerscan be inserted into target polynucleotides by removing the nucleobasesfrom one or more adjacent nucleotides. For instance, polunucleotides maybe modified to include 3-methyladenine, 7-methylguanine,1,N6-ethenoadenine inosine or hypoxanthine and the nucleobases may beremoved from these nucleotides using Human Alkyladenine DNA Glycosylase(hAAG). Alternatively, polunucleotides may be modified to include uraciland the nucleobases removed with Uracil-DNA Glycosylase (UDG). In oneembodiment, the one or more spacers do not comprise any abasicnuclcotides.

The one or more helicases may be stalled by (i.e. before) or on eachlinear molecule spacers. If linear molecule spacers are used, the targetpolynucleotide is preferably provided with a double stranded region ofpolynucleotide adjacent to the end of each spacer past which the one ormore helicases are to be moved. The double stranded region typicallyhelps to stall the one or more helicases on the adjacent spacer. Thepresence of the double stranded region(s) is particularly preferred ifthe method is carried out at at a salt concentration of about 100 mM orlower. Each double stranded region is typically at least 10, such as atleast 12, nucleotides in length. If the target polynucleotide used inthe invention is single stranded, a double stranded region may formed byhybridising a shorter polynucleotide to a region adjacent to a spacer.The shorter polynucleotide is typically formed from the same nucleotidesas the target polynucleotide, but may be formed from differentnucleotides. For instance, the shorter polynucleotide may be formed fromLNA.

If linear molecule spacers are used, the target polynucleotide ispreferably provided with a blocking molecule at end of each spaceropposite to end past which the one or more helicases are to be moved.This can help to ensure that the one or more helicases remain stalled oneach spacer. It may also help retain the one or more helicases on thetarget polynucleotide in the case that it/they diffuse(s) off insolution. The blocking molecule may be any of the chemical groupsdiscussed below which physically cause the one or more helicases tostall. The blocking molecule may be a double stranded region ofploynucleotide.

The one or more spacers preferably comprise one or more chemical groupswhich physically cause the one or more helicases to stall. The one ormore chemical groups are preferably one or more pendant chemical groups.The one or more chemical groups may be attached to one or morenucleobases in the target polynucleotide. The one or more chemicalgroups may be attached to the target polynucleotide backbone. Any numberof these chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more. Suitable groups include, but are not limited to,fluorophores, streptavidin and/or biotin, cholesterol, methylene blue,dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin anddibenzylcyclooctyne groups.

Different spacers in the target polynucleotide may comprise differentstalling molecules. For instance, one spacer may comprise one of thelinear molecules discussed above and another spacer may comprise one ormore chemical groups which physically cause the one or more helicases tostall. A spacer may comprise any of the linear molecules discussed aboveand one or more chemical groups which physically cause the one or morehelicases to stall, such as one or more abasics and a fluorophore.

Suitable spacers can be designed depending on the type of targetpolynucleotide and the conditions under which the method of theinvention is carried out. Most helicases bind and move along DNA and somay be stalled using anything that is not DNA. Suitable molecules arediscussed above.

The method of the invention is preferably carried out in the presence offree nucleotides and/or the presence of a helicase cofactor. This isdiscussed in more detail below. In the absence of the transmembrane poreand an applied potential, the one or more spacers are preferably capableof stalling the one or more helicases in the presence of freenucleotides and/or the presence of a helicase cofactor.

If the method of the invention is carried out in the presence of freenucleotides and a helicase cofactor as discussed below (such that theone of more helicases are in the active mode), one or more longerspacers are typically used to ensure that the one or more helicases arestalled on the target polynucleotide before they are contacted with thetransmembrane pore and a potential is applied. One or more shorterspacers may be used in the absence of free nucleotides and a helicasecofactor (such that the one or more helicases are in the inactive mode).

The salt concentration also affects the ability of the one or morespacers to stall the one or more helicases. In the absence of thetransmembrane pore and an applied potential, the one or more spacers arepreferably capable of stalling the one or more helicases at a saltconcentration of about 100 mM or lower. The higher the saltconcentration used in the method of the invention, the shorter the oneor more spacers that are typically used and vice versa.

Preferred combinations of features are shown in Table 1 below.

Spacer length (i.e. Target Spacer number Free Helicase polynucleotidecomposition* of *) Salt [ ] nucleotides? cofactor? DNA iSpC3 4 1M YesYes DNA iSp18 4  100-1000 mM Yes Yes DNA iSp18 6 <100-1000 mM Yes YesDNA iSp18 2 1M Yes Yes DNA iSpC3 12 <100-1000 mM Yes Yes DNA iSpC3 20<100-1000 mM Yes Yes DNA iSp9 6  100-1000 mM Yes Yes DNA idSp 4 1M YesYes

As discussed in more detail below, the method may concern moving two ormore helicases past a spacer. In such instances, the length of thespacer is typically increased to prevent the trailing helicase frompushing the leading helicasc past the spacer in the absence of the poreand applied potential. If the method concerns moving two or morehelicases past one or more spacers, the spacer lengths discussed abovemay be increased at least 1.5 fold, such 2 fold, 2.5 fold or 3 fold. Forinstance, if the method concerns moving two or more helicases past oneor more spacers, the spacer lengths in the third column of Table 1 abovemay be increased 1.5 fold, 2 fold, 2.5 fold or 3 fold.

The two or more helicases may also be separated such that each has itsown one or more spacers. This is discussed in more detail below.

Helicase(s)

Any helicase may be used in the invention. The helicase may be or bederived from a Hel308 helicase, a RecD helicase, such as TraI helicaseor a TrwC helicase, a XP helicase or a Dda helicase. The helicase may beany of the helicases, modified helicases or helicase constructsdisclosed in International Application Nos. PCT/GB2012/052579 (publishedas WO 2013/057495); PCT/GB2012/053274 (published as WO 2013/098562);PCT/0B2012/053273 (published as WO2013098561); PCT/GB2013/051925;PCT/GB2013/051924 and PCT/GB2013/051928; and in UK Application No.1318464.3 filed on 18 Oct. 2013).

The helicase preferably comprises the sequence shown in SEQ ID NO: 17(Trwe Cba) or as variant thereof, the sequence shown in SEQ ID NO: 28(Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 8(Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 8 comprises E94C/A360C and then(ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and 02).

Any number of helicases may be moved past the one or more spacers inaccordance with the invention. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more helicases may be moved past the one or more spacer. In someembodiments, different numbers of helicases may be moved past eachspacer. For instance, if two helicases are stalled using two separatespacers, one helicase (the first helicase) may be moved past the firstspacer, but two helicases (the first and second helicases) may be movedpast the second spacer.

The method of the invention preferably comprises moving two or more,such as three or more or four or more, stalled helicases past one ormore spacers. The two or more helicases are typically the samehelicases. The two or more helicases may be different helicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925;PCT/0B2013/051924 and PCT/GB2013/051928; and in UK Application No.1318464.3 filed on 18 Oct. 2013.

Conditions and Transmembrane Pores

The method comprises applying a potential across the pore. The appliedpotential may be a voltage potential. The method may comprise applying avoltage potential across the pore. The method may comprise increasingthe voltage applied across the pore. In this embodiment, the initialvoltage potential is typically not sufficient to move the one or morehelicases past the one or more spacer and the increased voltagepotential is typically sufficient to move the one or more helicases pastthe one or more spacers. Alternatively, the applied potential may be achemical potential. An example of this is using a salt gradient acrossan amphiphilic layer. A salt gradient is disclosed in Holden et al., JAm Chem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, thecurrent passing through the pore as the target polynucleotide moves withrespect to the pore is used to determine the sequence of the targetpolynucleotide. This is strand sequencing.

A transmembrane pore is a structure that crosses the membrane to somedegree. It permits hydrated ions driven by an applied potential to flowacross or within the membrane. The transmembrane pore typically crossesthe entire membrane so that hydrated ions may flow from one side of themembrane to the other side of the membrane. However, the transmembranepore does not have to cross the membrane. It may be closed at one end.For instance, the pore may be a well in the membrane along which or intowhich hydrated ions may flow.

Any transmembrane pore may be used in the invention. The pore may bebiological or artificial. Suitable pores include, but are not limitedto, protein pores, polynucleotide pores and solid state pores.

Any membrane may be used in accordance with the invention. Suitablemembranes are well-known in the art. The membrane is preferably anamphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have both at leastone hydrophilic portion and at least one lipophilic or hydrophobicportion. The amphiphilic molecules may be synthetic or naturallyoccurring. Non-naturally occurring amphiphiles and amphiphiles whichform a monolayer are known in the art and include, for example, blockcopolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450).Block copolymers are polymeric materials in which two or more monomersub-units that are polymerized together to create a single polymerchain. Block copolymers typically have properties that are contributedby each monomer sub-unit. However, a block copolymer may have uniqueproperties that polymers formed from the individual sub-units do notpossess. Block copolymers can be engineered such that one of the monomersub-units is hydrophobic (i.e. lipophilic), whilst the other sub-unit(s)are hydrophilic whilst in aqueous media. In this case, the blockcopolymer may possess amphiphilic properties and may form a structurethat mimics a biological membrane. The block copolymer may be a diblock(consisting of two monomer sub-units), but may also be constructed frommore than two monomer sub-units to form more complex arrangements thatbehave as amphipiles. The copolymer may be a triblock, tetrablock orpentablock copolymer.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically a planar lipid bilayer or a supported bilayer.

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers aremodels of cell membranes and serve as excellent platforms for a range ofexperimental studies. For example, lipid bilayers can be used for invitro investigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/0B08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Examples. Lipid bilayers are commonlyformed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA.,1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734).

In another preferred embodiment, the membrane is a solid state layer. Asolid-state layer is not of biological origin. In other words, a solidstate layer is not derived from or isolated from a biologicalenvironment such as an organism or cell, or a synthetically manufacturedversion of a biologically available structure. Solid state layers can beformed from both organic and inorganic materials including, but notlimited to, microelectronic materials, insulating materials such asSi₃N4, Al₂O₃, and SiO, organic and inorganic polymers such as polyamide,plastics such as Teflon® or elastomers such as two-componentaddition-cure silicone rubber, and glasses. The solid state layer may beformed from monatomic layers, such as graphene, or layers that are onlya few atoms thick. Suitable graphene layers are disclosed inInternational Application No. PCT/US2008/010637 (published as WO2009/035647).

The method is typically carried out using (i) an artificial amphiphiliclayer comprising a pore, (ii) an isolated, naturally-occurring lipidbilayer comprising a pore, or (iii) a cell having a pore insertedtherein. The method is typically carried out using an artificialamphiphilic layer, such as an artificial lipid bilayer. The layer maycomprise other transmembrane and/or intramembrane proteins as well asother molecules in addition to the pore. Suitable apparatus andconditions are discussed below. The method of the invention is typicallycarried out in vitro.

The target polynucleotide is preferably coupled to the membrane. Thismay be done using any known method. If the membrane is an amphiphiliclayer, such as a lipid bilayer (as discussed in detail above), thetarget polynucleotide is preferably coupled to the membrane via apolypeptide present in the membrane or a hydrophobic anchor present inthe membrane. The hydrophobic anchor is preferably a lipid, fatty acid,sterol, carbon nanotube or amino acid.

The target polynucleotide may be coupled directly to the membrane. Itmay be coupled to the membrane using any of the ways disclosed inInternational Application Number No. PCT/GB2012/051191 (published as WO2012/164270). The target polynucleotide is preferably coupled to themembrane via a linker. Preferred linkers include, but are not limitedto, polymers, such as polynucleotides, polyethylene glycols (PEGs) andpolypeptides. If a target polynucleotide is coupled directly to themembrane, then some data will be lost as the characterising run cannotcontinue to the end of the polynucleotide due to the distance betweenthe membrane and the pore and/or helicase. If a linker is used, then thetarget polynucleotide can be processed to completion. If a linker isused, the linker may be attached to the target polynucleotide at anyposition. The linker is typically attached to the target polynucleotideat the tail polymer.

The coupling may be stable or transient. For certain applications, thetransient nature of the coupling is preferred. If a stable couplingmolecule were attached directly to either the 5′ or 3′ end of apolynucleotide, then some data will be lost as the characterising runcannot continue to the end of the polynucleotide due to the distancebetween the membrane and the pore and/or helicase. If the coupling istransient, then when the coupled end randomly becomes free of themembrane, then the polynucleotide can be processed to completion.Chemical groups that form stable or transient links with the membraneare discussed in more detail below. The polynucleotide may betransiently coupled to an amphiphilic layer, such as a lipid bilayerusing cholesterol or a fatty acyl chain. Any fatty acyl chain having alength of from 6 to 30 carbon atoms, such as hexadecanoic acid, may beused.

In preferred embodiments, the polynucleotide is coupled to anamphiphilic layer. Coupling of polynucleotides to synthetic lipidbilayers has been carried out previously with various differenttethering strategies. These are summarised in Table 2 below.

TABLE 2 Attachment group Type of coupling Reference Thiol StableYoshina-lshii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol- based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Lipid Stable van Lengerich,B., R. J. Rawle, et al. “Covalent attachment of lipid vesicles to afluid-supported bilayer allows observation of DNA-mediated vesicleinteractions.” Langmuir 26(11): 8666-72

Polynucleotides may be functionalized using a modified phosphoramiditein the synthesis reaction, which is easily compatible for the additionof reactive groups, such as thiol, cholesterol, lipid and biotin groups.These different attachment chemistries give a suite of attachmentoptions for polynucleotides. Each different modification group tethersthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the membrane. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides can also be achieved by a number of othermeans provided that a reactive group can be added to the polynucleotide.The addition of reactive groups to either end of DNA has been reportedpreviously. A thiol group can be added to the 5′ of ssDNA usingpolynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “Afacile method for attaching nitroxide spin labels at the 5′ terminus ofnucleic acids.” Nucleic Acids Res 35(10): e77). A more diverse selectionof chemical groups, such as biotin, thiols and fluorophores, can beadded using terminal transferase to incorporate modifiedoligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tehen, et al. (1988).“Nonradioactive labeling of synthetic oligonucleotide probes withterminal deoxynucleotidyl transferase.” Anal Biochem 169(2): 376-82).

Alternatively, the reactive group could be considered to be a shortregion in the polynucleotide to one already coupled to the membrane, sothat attachment can be achieved via hybridisation. The region could bepart of the polynucleotide or ligated to it. Ligation of short pieces ofssDNA have been reported using T4 RNA ligase I (Troutt, A. B., M. G.McHeyzer-Williams, et al. (1992). “Ligation-anchored PCR: a simpleamplification technique with single-sided specificity.” Proc Natl AcadSci USA 89(20): 9823-5).

Most preferably, the polynucleotide is coupled to the membrane using acholesterol-tagged polynucleotide which hybridises to thepolynucleotide.

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as analyte, to flow fromone side of a membrane to the other side of the membrane. In the presentinvention, the transmembrane protein pore is capable of forming a porethat permits hydrated ions driven by an applied potential to flow fromone side of the membrane to the other. The transmembrane protein porepreferably permits analyte such as nucleotides to flow from one side ofthe membrane, such as a lipid bilayer, to the other. The transmembraneprotein pore allows a polynucleotide or nucleic acid, such as DNA orRNA, to be moved through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as 6, 7, 8 or9 subunits. The pore is preferably a hexameric, heptameric, octameric ornonameric pore.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane ca-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, lysening, outer membraneporin F (OmpF), outer membrane porin G (OmpG), outer membranephospholipase A and Netsseria autotransporter lipoprotein (NalP).α-helix bundle pores comprise a barrel or channel that is formed fromα-helices. Suitable α-helix bundle pores include, but are not limitedto, inner membrane proteins and a outer membrane proteins, such as WZAand CIyA toxin. The transmembrane pore may be derived from lysenin.Suitable pores derived from lysenin are disclosed in InternationalApplication No. PCT/GB2013/050667 (published as WO 2013/153359). Thetransmembrane pore may be derived from Msp or from α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp,preferably from MspA. Such a pore will be oligomeric and typicallycomprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be ahomo-oligomeric pore derived from Msp comprising identical monomers.Alternatively, the pore may be a hetero-oligomeric pore derived from Mapcomprising at least one monomer that differs from the others. Preferablythe pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp typically comprises the sequence shown in SEQID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant ofthe MspA monomer. It includes the following mutations: D90N, D91N, D93N,D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide thathas an amino acid sequence which varies from that of SEQ ID NO: 2 andwhich retains its ability to form a pore. The ability of a variant toform a pore can be assayed using any method known in the art. Forinstance, the variant may be inserted into an amphiphilic layer alongwith other appropriate subunits and its ability to oligomerise to form apore may be determined. Methods are known in the art for insertingsubunits into membranes, such as amphiphilic layers. For example,subunits may be suspended in a purified form in a solution containing alipid bilayer such that it diffuses to the lipid bilayer and is insertedby binding to the lipid bilayer and assembling into a functional state.Alternatively, subunits may be directly inserted into the membrane usingthe “pick and place” method described in M. A. Holden, H. Bayley. J. Am.Chem. Soc. 2005, 127, 6502-6503 and International Application No.PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant may be atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% and more preferably at least 95%,97% or 99% homologous based on amino acid identity to the amino acidsequence of SEQ ID NO: 2 over the entire sequence. There may be at least80%0, for example at least 85%, 90% or 95%, amino acid identity over astretch of 100 or more, for example 125, 150, 175 or 200 or more,contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altachul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7.In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N102E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of 01, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, 149H, I68V,D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B, C and D. The variant preferably comprises the mutation L88N.A variant of SEQ ID NO: 2 has the mutation L88N in addition to all themutations of MS-(B1)8 and is called MS-(B2)8. The pore used in theinvention is preferably MS-(B2)8. The further preferred variantcomprises the mutations G75S/G77S/L88N/Q126R. The variant of SEQ ID NO:2 has the mutations G75S/G77S/L88N/Q126R in addition to all themutations of MS-(B1)8 and is called MS-(B2C)8. The pore used in theinvention is preferably MS-(B2)8 or MS-(B2C)8.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 3below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 4.

TABLE 3 Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Ashpolar, hydrophilic, neutral Asp polar, hydrophilic, charged (−) Prohydrophobic, neutral Glu polar, hydrophilic, charged (−) Gln polar,hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar,hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, hydrophilic, Thr polar, hydrophilic,charged (+) neutral Ile aliphatic, hydrophobic, neutral Val aliphatic,hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic,hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

TABLE 4 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the sequence of SEQ ID NO: 2 mayadditionally be deleted from the polypeptides described above. Up to 1,2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form 1-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a his tag), aspartic acid residues (an asp tag), astreptavidin tag or a flag tag, or by the addition of a signal sequenceto promote their secretion from a cell where the polypeptide does notnaturally contain such a sequence. An alternative to introducing agenetic tag is to chemically react a tag onto a native or engineeredposition on the pore. An example of this would be to react a gel-shiftreagent to a cysteine engineered on the outside of the pore. This hasbeen demonstrated as a method for separating hemolysin hetero-oligomers(Chem Biol. 1997 July; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. Therevealing label may be any suitable label which allows the pore to bedetected. Suitable labels are described below.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the pore may be synthesized by invitro translation and transcription (IVTT). Suitable methods forproducing pores are discussed in International Application Nos.PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived fromα-hemolysin (α-HL). The wild type α-HL pore is formed of seven identicalmonomers or subunits (i.e. it is heptameric). The sequence of onemonomer or subunit of α-hemolysin-NN is shown in SEQ ID NO: 4. Thetransmembrane protein pore preferably comprises seven monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof.Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217,218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4form part of a constriction of the barrel or channel of α-HL.

In such embodiments, a pore comprising seven proteins or monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof arepreferably used in the method of the invention. The seven proteins maybe the same (homo-heptamer) or different (hetero-heptamer).

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 4 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into an amphiphilic layer, such as a lipid bilayer, along withother appropriate subunits and its ability to oligomerise to form a poremay be determined. Methods are known in the art for inserting subunitsinto amphiphilic layers, such as lipid bilayers. Suitable methods arediscussed above.

The variant may include modifications that facilitate covalentattachment to or interaction with the helicase. The variant preferablycomprises one or more reactive cysteine residues that facilitateattachment to the helicasc. For instance, the variant may include acysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51,237, 239 and 287 and/or on the amino or carboxy terminus of SEQ ID NO:4. Preferred variants comprise a substitution of the residue at position8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (A8C, T9C,N17C, K237C, S239C or E287C). The variant is preferably any one of thevariants described in International Application No. PCT/GB09/001690(published as WO 2010/004273), PCT/GB09/001679 (published as WO2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).

The variant may also include modifications that facilitate anyinteraction with nucleotides.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium.Alternatively, the variant may be expressed in vitro or recombinantly bya bacterium such as Escherichia coli. Variants also includenon-naturally occurring variants produced by recombinant technology.Over the entire length of the amino acid sequence of SEQ ID NO: 4, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 4 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 4 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:4 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 4. Such fragments retainpore-forming activity. Fragments may be at least 50, 100, 200 or 250amino acids in length. A fragment preferably comprises the pore-formingdomain of SEQ ID NO: 4. Fragments typically include residues 119, 121,135. 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 4 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 4 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a β-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 4 typically comprisesthe regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQID NO: 4 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 4 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the β-strand regions ofSEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

The variant may be modified to assist its identification or purificationas discussed above.

Pores derived from α-HL can be made as discussed above with reference topores derived from Map.

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. The transmembrane protein pore is preferably chemically modifiedby attachment of a molecule to one or more cysteines (cysteine linkage),attachment of a molecule to one or more lysines, attachment of amolecule to one or more non-natural amino acids, enzyme modification ofan epitope or modification of a terminus. Suitable methods for carryingout such modifications are well-known in the art. The transmembraneprotein pore may be chemically modified by the attachment of anymolecule. For instance, the pore may be chemically modified byattachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers ispreferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may beattached directly to the pore or attached via a linker as disclosed inInternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

The helicase may be covalently attached to the pore. The helicase ispreferably not covalently attached to the pore.

Any of the proteins described herein may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a his tag), aspartic acid residues (an asp tag), astreptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or bythe addition of a signal sequence to promote their secretion from a cellwhere the polypeptide does not naturally contain such a sequence. Analternative to introducing a genetic tag is to chemically react a tagonto a native or engineered position on the helicase or pore. An exampleof this would be to react a gel-shift reagent to a cysteine engineeredon the outside of the pore. This has been demonstrated as a method forseparating hemolysin hetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

The target polynucleotide, helicase or pore may be labelled with arevealing label. The revealing label may be any suitable label which canbe detected. Suitable labels include, but are not limited to,fluorescent molecules, radioisotopes, e.g. ¹²⁵I, ^(3S)S, enzymes,antibodies, antigens, polynucleotides and ligands such as biotin.

Proteins may be made synthetically or by recombinant means. For example,proteins may be synthesized by in vitro translation and transcription(IVTT). The amino acid sequence of the protein may be modified toinclude non-naturally occurring amino acids or to increase the stabilityof the protein. When a protein is produced by synthetic means, suchamino acids may be introduced during production. Proteins may also bealtered following either synthetic or recombinant production.

Proteins may also be produced using D-amino acids. For instance, thepore or helciase may comprise a mixture of L-amino acids and D-aminoacids. This is conventional in the art for producing such proteins orpeptides.

The proteins used in the invention may also contain other non-specificmodifications as long as they do not interfere with the proteins'function. A number of non-specific side chain modifications are known inthe art and may be made to the side chains of the protein(s). Suchmodifications include, for example, reductive alkylation of amino acidsby reaction with an aldehyde followed by reduction with NaBH₄,amidination with methylacetimidate or acylation with acetic anhydride.

Polynucleotide sequences encoding a protein may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a protein may be expressed in a bacterial host cell usingstandard techniques in the art. The protein may be produced in a cell byin situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

The gene encoding the sequence of interest may be amplified using PCRinvolving specific primers. The amplified sequences may then beincorporated into a recombinant replicable vector such as a cloningvector. The vector may be used to replicate the polynucleotide in acompatible host cell. Thus polynucleotide sequences may be made byintroducing a polynucleotide encoding the sequence of interest into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells for cloning of polynucleotides are known in the art anddescribed in more detail below.

The polynucleotide sequence may be cloned into a suitable expressionvector. In an expression vector, the polynucleotide sequence istypically operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by the host cell.Such expression vectors can be used to express a construct.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotide maybe introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a construct can be produced by inserting a polynucleotide sequenceencoding a construct into an expression vector, introducing the vectorinto a compatible bacterial host cell, and growing the host cell underconditions which bring about expression of the polynuclcotide sequence.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,ara or λ_(L) promoter is typically used.

The host cell typically expresses the construct at a high level. Hostcells transformed with a polynucleotide sequence will be chosen to becompatible with the expression vector used to transform the cell. Thehost cell is typically bacterial and preferably E. coli. Any cell with aλ DE3 lysogen, for example Rosetta2(DE3)pLys, C41 (DE3), BL21 (DE3),JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express avector comprising the T7 promoter.

Proteins may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

The method of the invention involves measuring one or morecharacteristics of the target polynucleotide. The method may involvemeasuring two, three, four or five or more characteristics of the targetpolynucleotide. The one or more characteristics are preferably selectedfrom (i) the length of the target polynucleotide, (ii) the identity ofthe target polynucleotide, (iii) the sequence of the targetpolynucleotide, (iv) the secondary structure of the targetpolynucleotide and (v) whether or not the target polynucleotide ismodified. Any combination of (i) to (v) may be measured in accordancewith the invention.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the target polynucleotideand the pore or the duration of interaction between the targetpolynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the targetpolynucleotide or without measurement of the sequence of the targetpolynucleotide. The former is straightforward; the polynucleotide issequenced and thereby identified. The latter may be done in severalways. For instance, the presence of a particular motif in thepolynucleotide may be measured (without measuring the remaining sequenceof the polynucleotide). Alternatively, the measurement of a particularelectrical and/or optical signal in the method may identify the targetpolynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not the targetpolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcytosine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunnelling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11(1):279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined withelectrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January;81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO-2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International ApplicationWO-2009/077734 and International Application WO-2011/067559.

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore is present in amembrane. The method may be carried out using any apparatus that issuitable for transmembrane pore sensing. For example, the apparatuscomprises a chamber comprising an aqueous solution and a barrier thatseparates the chamber into two sections. The barrier typically has anaperture in which the membrane containing the pore is formed.Alternatively the barrier forms the membrane in which the pore ispresent.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the poreas the polynucleotide moves with respect to the pore. Therefore theapparatus may also comprise an electrical circuit capable of applying apotential and measuring an electrical signal across the membrane andpore. The methods may be carried out using a patch clamp or a voltageclamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExamples. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +2 V to−2 V, typically −400 mV to +400 mV. The voltage used is preferably in arange having a lower limit selected from −400 mV, −300 mV, −200 mV, −150mV, −100 mV, −50 mV, −20 mV and 0 mV and an upper limit independentlyselected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mVand +400 mV. The voltage used is more preferably in the range 100 mV to240 mV and most preferably in the range of 120 mV to 220 mV. It ispossible to increase discrimination between different nucleotides by apore by using an increased applied potential.

The methods are typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The salt concentration may be at saturation.The salt concentration may be 3 M or lower and is typically from 0.1 to2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9to 1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from150 mM to 1 M. Hel308, XPD, RecD, TraI and Dda helicases surprisinglywork under high salt concentrations. The method is preferably carriedout using a salt concentration of at least 0.3 M, such as at least 0.4M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, atleast 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High saltconcentrations provide a high signal to noise ratio and allow forcurrents indicative of the presence of a nucleotide to be identifiedagainst the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports helicase function, such as about 37° C.

The method may be carried out in the presence of free nucleotides orfree nucleotide analogues and/or a helicase cofactor that facilitatesthe action of the helicase. The method may also be carried out in theabsence of free nucleotides or free nucleotide analogues and in theabsence of a helicase cofactor. The free nucleotides may be one or moreof any of the individual nucleotides discussed above. The freenucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The free nucleotides are preferably selected from AMP, TMP, GMP, CMP,UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferablyadenosine triphosphate (ATP). The helicase cofactor is a factor thatallows the helicase or construct to function. The helicase cofactor ispreferably a divalent metal cation. The divalent metal cation ispreferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The helicase cofactor is mostpreferably Mg²⁺.

Methods of Controlling the Loading of One or More Helicases on a TargetPolynucleotide

The invention also provides a method of controlling the loading of oneor more helicases on a target polynucleotide. The method comprisesproviding the target polynucleotide with one or more spacers. The methodpreferably comprises modifying the target polynucleotide so that itcomprises one or more spacers. All of the spacer embodiments discussedabove equally apply to this method.

The method also comprises contacting the target polynucleotide with theone or more helicases such that the one or more helicases bind to thetarget polynucleotide and one or more helicases stall at each spacer.The stalling of helicases at spacers may be assayed as discussed above.

The target polynucleotide may comprise any number of spacers asdiscussed above. The target polynucleotide preferably comprises two ormore spacers, such as 3, 4, 5, 6, 7, 8, 9, 10 or more spacers. Anynumber of helicases may be stalled at each spacer as discussed above. Inthis way, it is possible to control where and how many helicases areloaded on the target polynucleotide and thereby facilitatecharacterisation of the target polynucleotide. The one or more helicasesmay be moved past the one or more spacers using any of the methodsdiscussed above.

The target polynucleotide is preferably provided with one or morespacers S and one or more single stranded regions or one or morenon-hybridised regions L (L is for loading site). The length of eachregion L depends on the number of helicases that should bind to each Land be stalled at each spacer S. The one or more spacers S and one ormore regions L may be adjacent to (i.e. next to) one another or may beseparated by part of the target polynucleotide. Each spacer is typicallylocated at or near the end of each region L towards which the helicasemoves. For instance, if the helicase is a 5′ to 3′ helicase, each spaceris typically located at or near the 3′ end of each region, i.e5′-L-S-3′. If the helicase is a 3′ to 5′ helicase, each spacer istypically located at or near the 5′ end of each region, i.e 5′-S-L-3′.

The target polynucleotide is preferably provided with (L-S)n or (S-L)nin the 5′ to 3′ direction, wherein L is a single stranded polynucleotideor a non-hybridised polynucleotide, S is a spacer and n is a wholenumber, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. n is preferably1, 2, 3 or 4. The 5′ to 3′ direction refers to the targetpolynucleotide.

The target polynucleotide is preferably provided with one or more singlestranded regions or one or more non-hybridised regions L each of whichhas a spacer S at or near either end, i.e. provided with (S-L-S)n.

In a preferred embodiment, the spacer is adjacent to a double strandedregion D as discussed above, i.e. 5′-L-S-D-3′ for 5′ to 3′ helicases orhelicases used in the inactive mode or 5′-D-S-L-3′ for 3′ to 5′helicases or helicases used in the inactive mode. The targetpolynucleotide is preferably provided with (L-S-D)n or (D-S-L)n in the5′ to 3′ direction, wherein L is a single stranded polynucleotide or anon-hybridised polynucleotide, S is a spacer, D is a double strandedpolynucleotide and n is a whole number, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more. n is preferably 1, 2, 3 or 4. L may be the same type ofpolynucleotide as D or may be a different type of polynucleotide from D.L and/or D may be the same type of polynucleotide as the targetpolynucleotide or may be a different type of polynucleotide from thetarget polynucleotide.

In a preferred embodiment, a blocking molecule B is provided at the endof each spacer opposite to the end past which the one or more helicasesare to be moved, i.e. 5′-B-L-S-3′ for 5′ to 3′ helicases or helicasesused in the inactive mode or 5′-S-L-B-3′ for 3′ to 5′ helicases. Thetarget polynucleotide is preferably provided with (B-L-S)n or (S-L-B)nin the 5′ to 3′ direction, wherein L is a single stranded polynucleotideor a non-hybridised polynucleotide, S is a spacer, B is blockingmolecule and n is a whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more. n is preferably 1, 2, 3 or 4.

In the most preferred embodiment, the target polynucleotide is providedwith both D and B, i.e 5′-B-L-S-D-3′ for 5′ to 3′ helicases or helicasesused in the inactive mode or 5′-D-S-L-B-3′ for 3′ to 5′ helicases. Thetarget polynucleotide is most preferably provided with (B-L-S-D)n or(D-S-L-B)n in the 5′ to 3′ direction, wherein L is a single strandedpolynucleotide or a non-hybridised polynucleotide, S is a spacer, B isblocking molecule, D is a double stranded polynucleotide and n is awhole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. n ispreferably 1, 2, 3 or 4.

The target polynucleotide may be provided with any number of thesespacer-containing units. For instance, the target polynucleotide may beprovided with (5′-L-S-3′)n, (5′-S-L-3′)n, (S-L-S)n, (5′-L-S-D-3′)n,(5′-D-S-L-3′)n, (5′-B-L-S-3′)n, (5′-S-L-B-3′)n, (5′-B-L-S-D-3′)n or(5′-D-S-L-B-3′)n, where n is 2 or more, such as such as 3, 4, 5, 6, 7,8, 9, 10 or more. Such embodiments allow multiple helicases to bestalled on the target polynucleotide.

The target polynucleotide may be provided with all of the embodimentsdiscussed above with reference to L, S, D and B by ligating an adaptorof the invention to the target polynucleotide.

In a preferred embodiment, the target polynucleotide is contacted withthe one or more helicases such that one helicase (i.e. only onehelicase) stalls at each spacer. This can be achieved by providing thetarget polynucleotide with one or more spacers S and one or more singlestranded regions or one or more non-hybridised regions L¹ each of whichis only long enough for one helicase to bind. The target polynucleotideis preferably provided with (L¹-S)n or (S-L¹)n in the 5′ to 3′direction, wherein L¹ is a single stranded polynucleotide or anon-hybridised polynucleotide which is only long enough for one helicaseto bind, S is a spacer and n is a whole number, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more. n is preferably 1, 2, 3 or 4.

The length of region L¹ depends on the footprint of the helicase and canbe calculated in a straightforward manner. Region L may be part of thetarget polynucleotide or may be added to the target polynucleotide, forinstance as part of an adaptor of the invention. Region L¹ is typically8, 9, 10, 12, 13, 14 or 15 nucleotides in length. The one or morespacers S and one or more L¹ regions may be adjacent to (i.e. next to)one another or may be separated by part of the target polynucleotide.Each spacer S is typically located at or near the end of each region L¹towards which the helicase moves. For instance, if the helicase is a 5′to 3′ helicase, each spacer S is typically located at or near the 3′ endof each region L¹, i.e 5′-L¹-S-3′. If the helicase is a 3′ to 5′helicase, each spacer S is typically located at or near the 5′ end ofeach region L¹, i.e 5′-S-L¹-3′. The target polynucleotide is preferablyprovided with one or more single stranded regions or one or morenon-hybridised regions L¹ each of which is only long enough for onehelicase to bind and each of which has a spacer S at or near either end,i.e. (S-L¹-S)n.

The target polynucleotide may be provided with (5′-L¹-S-3′)n,(5′-S-L¹-3′)n, (S-L¹-S)n, (5′-L¹-S-D-3′)n, (5′-D-S-L-3′)n,(5′-B-L¹-S-3′)n, (5′-S-L¹-B-3′)n, (5′-B-L¹-S-D-3′)n or(5′-D-S-L¹-B-3′)n, where n is a whole number, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more. n is preferably 1, 2, 3 or 4. Such embodimentsallow n helicases to be stalled on the target polynucleotide. Onehelicase is stalled by each spacer.

In another preferred embodiment, the target polynucleotide is contactedwith the one or more helicases such that two helicases (i.e. only twohelicases) stall at each spacer. This can be achieved by providing thetarget polynucleotide with one or more spacers S and one or more singlestranded regions or one or more non-hybridised regions L² each of whichis only long enough for two helicases to bind. The target polynucleotideis preferably provided with (L²-S)n or (S-L²)n in the 5′ to 3′direction, wherein L is a single stranded polynucleotide or anon-hybridised polynucleotide which is only long enough for twohelicases to bind, S is a spacer and n is a whole number, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more. n is preferably 1, 2, 3 or 4.

The length of region L² depends on the footprint of the helicases andcan be calculated in a straightforward manner. Region L² may be part ofthe target polynucleotide or may be added to the target polynucleotide,for instance as part of an adaptor of the invention. Region L² istypically 16, 17, 18, 19, 20, 21 or 22 nucleotides in length. The one ormore spacers S and one or more regions L² may be adjacent to (i.e. nextto) one another or may be separated by part of the polynucleotide. Eachspacer is typically located at or near the end of each region towardswhich the helicase moves. For instance, if the helicase is a 5′ to 3′helicase, each spacer is typically located at or near the 3′ end of eachregion. The polynucleotide is preferably provided with one or moresingle stranded regions or one or more non-hydrisied regions L² each ofwhich is only long enough for two helicases to bind and each of whichhas a spacer S at or near either end, i.e. (S-L²-S)n.

The target polynucleotide may be provided with (5′-L²-S-3′)n,(5′-S-L²-3′)n, (S-L²-S)n, (5′-L²-S-D-3′)n, (5′-D-S-L²-3′)n,(5′-B-L²-S-3′)n, (5′-S-L²-B-3′)n, (5′-B-L²-S-D-3′)n or(5′-D-S-L²-B-3′)n, where n is a whole number, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more. n is preferably 1, 2, 3 or 4. Such embodimentsallow 2n helicases to be stalled on the target polynucleotide. Twohelicases are stalled by each spacer.

The two helicases stalled at each spacer are preferably different fromone another. This can be controlled in several ways. For instance, twodifferent helicases may be attached to one another, such as covalentlyattached to one another, and then stalled at each spacer. Suitableconstructs are discussed above. Alternatively, blocking polynucleotidesmay be used to ensure that different helicases are stalled by eachspacer. If the method comprises providing the polynucleotide with one ormore spacers S and one or more single stranded regions or one or morenon-hydrisied regions L² each of which is only long enough for twohelicases to bind, the method preferably comprises hybridising ablocking polynucleotide to part of each region L² so that the remaining(i.e. non-blocked) part of each region is only long enough to bind onehelicase. Blocking polynucleotides are typically 2, 3, 4, 5, 6, 7 or 8nucleotides in length. The blocking polynucleotide prevents twohelicases from binding to the same region at the same time. Thepolynucleotide comprising the blocking polynucleotides is preferablycontacted with one or more helicases such that one helicase binds to theremaining (i.e. non-blocked) part of each region L². Each helicase maythen be used to remove each blocking polynucleotide. The one or morebound helicases are preferably provided with free nucleotides and ahelicase cofactor such that they remove each blocking polynucleotide andstall at each spacer S. The polynucleotide produced in in this way isthen preferably contacted with one or more helicases which are differentfrom the helicases used earlier in the method such that one differenthelicase binds to each region and is stalled by the spacer and the otherstalled helicase.

The method preferably comprises (a) providing the target polynucleotidewith (L²-S)n or (S-L²)n in the 5′ to 3′ direction, wherein L is a singlestranded polynucleotide or a non-hybridised polynucleotide which is onlylong enough for two helicases to bind, S is a spacer and n is a wholenumber, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more; (b) hybridising ablocking polynucleotide to part of each region L² so that the remainingpart of each region L² is only long enough to bind one helicase; (c)contacting the target polynucleotide produced in (b) with one or morehelicases such that one helicase binds to the remaining part of eachregion L²; (d) providing the one or more bound helicases in (c) withfree nucleotides and a helicase cofactor such that they remove eachblocking polynucleotide and stall at each spacer S; and (e) contactingthe target polynucleotide produced in (d) with one or more helicaseswhich are different from those used in (c) such that one differenthelicase binds to each region L² and is stalled by each spacer and eachhelicase stalled in (d). n is preferably 1, 2, 3 or 4. Otherarrangements of S and L², such (S-L²-S)n, (5′-L²-S-D-3′)n,(5′-D-S-L²-3′)n, (5′-B-L²-S-3′)n, (5′-S-L²-B-3′)n, (5′-B-L²-S-D-3′)n and(5′-D-S-L²-B-3′)n as discussed above, may also be used in thisembodiment.

As discussed above, the length of a spacer may be used to control thenumber of helicases that are stalled and/or the number of helicaseswhich may be moved past the spacer. Longer spacers may be used to stallmore helicases. Trains of two or more helicases, such as 3, 4 or 5helicases, may also move past longer spacers because trailing helicasesmay push leading helicases past the spacer. The embodiments withreference to L¹ and L² above can be modified such that 3, 4 or 5helicases are stalled at each spacer. For instance, the polynucleotidemay be provided with one or more spacers S and one or more singlestranded regions or one or more non-hybridised regions each of which isonly long enough for three (L3), four (L⁴) or five (L⁵) helicases tobind.

Adaptor

The invention also provides an adaptor for controlling the movement of atarget polynucleotide. The adaptor is preferably for characterising atarget polynucleotide. The adaptor comprises (a) (L-S-D)n or (D-S-L)n inthe 5′ to 3′ direction, wherein L is a single stranded polynucleotide ora non-hybridised polynucleotide, S is a spacer and D is a doublestranded polynucleotide and wherein n is a whole number, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more, and (b) one or more helicases stalledon each adaptor. n is preferably 1, 2, 3 or 4. The 5′ to 3′ directionrefers to the direction of the L and D polynucleotides in the adaptor.

The one or more helicases may be stalled before the spacer S, by thespacer S or on the spacer S.

The adaptor may be ligated to a target polynucleotide such that thetarget polynucleotide may be used in any of the method discussed above.

L may be L¹ or L² as discussed above. An adaptor may comprise acombination of L¹ and L².

All of the spacer embodiments discussed above equally apply to thismethod.

Any of the embodiments discussed above with reference to L, S and Dequally apply to the adaptors of the invention. The adaptor may comprise(5′-L¹-S-D-3′)n, (5′-D-S-L¹-3′)n, (5′-B-L¹-S-D-3′)n or(5′-D-S-L¹-B-3′)n, (5′-L²-S-D-3′)n, (5′-D-S-L²-3′)n, (5′-B-L²-S-D-3′)nor (5′-D-S-L²-B-3′)n in the 5′ to 3′ direction, where n is a wholenumber, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. n is preferably1, 2, 3 or 4. L¹ or L² may be replaced with L³, L⁴ or L⁵.

Most preferably n is 1 and one or two helicases are stalled one theadaptor.

Kit

The invention also provides a kit for controlling the movement of atarget polynucleotide. The kit is preferably for characterising a targetpolynucleotide. The kit comprises (a) one or more spacers, (b) one ormore helicases and (c) a transmembrane pore. All of the spacerembodiments discussed above with reference to the methods of theinvention equally apply to the kits of the invention. For instance, theone or more spacers may be part of a polynucleotide adaptor, preferablya single stranded polynucleotide adaptor, which may be ligated to to thetarget polynucleotide and which comprises a leader sequence whichpreferentially threads into the pore. The kit may comprise any of thehelicases and pores discussed above.

The one or more spacers and the one or more helicases may be part of anadaptor of the invention.

The kit may further comprise the components of a membrane, such as thephospholipids needed to form an amphiphilic layer, such as a lipidbilayer.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides, amembrane as defined above or voltage or patch clamp apparatus. Reagentsmay be present in the kit in a dry state such that a fluid sampleresuspends the reagents. The kit may also, optionally, compriseinstructions to enable the kit to be used in the method of the inventionor details regarding which patients the method may be used for. The kitmay, optionally, comprise the components necessary to facilitatehelicase movement (e.g. ATP and Mg²⁺). The following Examples illustratethe invention.

EXAMPLES Example 1

This example describes how a T4 Dda-E94C/A360C (SEQ ID NO: 8 withmutations E94C/A360C and then (ΔM1)G1G2) helicase can control themovement of intact DNA strands through a single MspA nanopore(MS(B1-G75S/G77S/L88N/Q126R)8 (MspA-B2C) (SEQ ID NO: 2 with mutationsG75S/G77S/L88N/Q126R). The iSpC3 spacers in the lambda DNA construct(SEQ ID NO: 9 attached by its 3′ end to four iSpC3 spacers which areattached to the 5′ end of SEQ ID NO: 10 which is attached to three iSpC3spacers which are attached to the 3′ end to SEQ ID NO: 11, the SEQ IDNO: 10 region of this construct is hybridised to SEQ ID NO: 12 (whichhas attached to its 3′ end, six iSp18 spacers attached to two thymineresidues and a 3′ cholesterol TEG)) are used to stall the enzyme untilthe construct is captured by the nanopore. Upon capture the force of theapplied potential moves the enzyme T4 Dda-E94C/A360C past the stallingspacer and allows enzyme controlled DNA movement of the lambda constructthrough the nanopore.

Materials and Methods

Prior to setting up the experiment, the Lambda DNA construct (SEQ ID NO:9 attached by its 3′ end to four iSpC3 spacers which are attached to the5′ end of SEQ ID NO: 10 which is attached at its 3′ end to SEQ ID NO:11, the SEQ ID NO: 10 region of this construct is hybridised to SEQ IDNO: 12 (which has a 3′ cholesterol tether)) and T4 Dda-E94C/A360C werepre-incubated together for 15 minutes at 23° C. in buffer (20 mM CAPS,pH 10.0, 500 mM NaCl, 5% Glycerol, 2 mM DTT).

Electrical measurements were acquired at 20° C. (by placing theexperimental system on a cooler plate) from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer (600 mM KCl, 25 mMpotassium phosphate, 75 mM Potassium Ferrocyanide (II), 25 mM Potassiumferricyanide (III), pH 8). After achieving a single pore inserted in theblock co-polymer, then buffer (1 mL, 600 mM KCl, 25 mM potassiumphosphate, 75 mM Potassium Ferrocyanide (II), 25 mM Potassiumferricyanide (III), pH 8) was flowed through the system to remove anyexcess MspA nanopores (MspA-B2C) and finally experimental buffer wasflowed into the system (2 mL 960 mM KCl, 25 mM potassium phosphate, 3 mMPotassium Ferrocyanide (II), 1 mM Potassium ferricyanide (HI), pH 8).MgCl₂ (10 mM final concentration) and ATP (1 mM final concentration)were mixed together with buffer (960 mM KCl, 25 mM potassium phosphate,3 mM Potassium Ferrocyanide (II), 1 mM Potassium ferricyanide (III), pH8) and then added to the Lambda DNA construct (0.2 nM finalconcentration), T4 Dda-E94C/A360C (10 nM final concentration) buffer (20mM CAPS, pH 10.0, 500 mM NaCl, 5% Glycerol, 2 mM DTT) pre-mix. Thepre-mix was then added to the single nanopore experimental system.Experiments were carried out for four hours following a potential flipprocess (+100 mV for 2 s, then 0 V for 2 s, then −120 mV for 14500sapplied at the cis side) and helicase-controlled DNA movement wasmonitored.

Results and Discussion

The DNA construct is shown in FIG. 1. The T4 Dda-E94C/A360C is able tobind on to the region of the construct labelled A (SEQ ID NO: 9) whenpre-incubated with the DNA construct. However, the enzyme is not able tomotor past the stalling groups when in free solution (labelled B and Din FIG. 1). Therefore, the enzyme is stalled at the iSpC3 spacers(labelled B in FIG. 1) until the DNA construct is captured by thenanopore. Once captured by the nanopore, the force of the appliedpotential moves the enzyme T4 Dda-E94C/A360C past the stalling spacerand allows enzyme controlled DNA movement of the lambda constructthrough the nanopore. An example of a helicase-controlled DNA movementis shown in FIG. 2. The helicase-controlled DNA movement was 5170seconds long and corresponds to the translocation of approximately 30 kBof the lambda construct through the nanopore. FIG. 3 shows zoomed inregions of the beginning (a) and end (b) of the helicase-controlled DNAmovement, 1 and 2 show when the iSpC3 spacers translocate through thenanopore under control of the helicase.

Example 2

The DNA construct used in this example was produced by fragmentation ofLambda DNA into ˜5-10 kB fragments using MuA. The fragments which wereproduced by the sample prep were then passed through a nanopore, withtheir movement controlled by a helicase enzyme. The helicase was movedpast the dsDNA region (where the tether hybridises to the construct) andthe spacers by the force of the applied potential across the nanopore.The observance of characteristic blocks produced by the helicasecontrolled movement of the markers through the nanopore showed thesample preparation procedure had been successful and that the enzyme hadbeen stalled as shown in FIG. 4 (b). This meant that the enzyme had adefined start point and was unable to move past the dsDNA region (wherethe tether hybridises to the construct) and spacers until captured bythe nanopore.

Materials and Methods 2.1 Anneal of DNA Strands to Form Y-Shaped andHairpin MuA Substrates

The Y-shaped and hairpin MuA substrates were prepared as shown in Table5 below. The sample mixtures which contained the DNA to form theY-shaped and hairpin MuA substrates were then heated to 95° C. for 2minutes and then cooled to 16° C. at a rate of 2° C. per minute. Thisallowed SEQ ID NOs: 14 and 15 (where SEQ ID NO: 14 is attached at its 3′end to the 5′ end of SEQ ID NO: 15 by four iSpC3 spacer units) to annealto SEQ ID NO: 19 and 9 (where SEQ ID NO: 19 is attached at its 3′ end tothe 5′ end of SEQ ID NO: 9 by four iSpC3 spacer units) to form theY-shaped MuA substrate and for SEQ ID NO: 19 and 20 (where SEQ ID NO: 19is attached at its 3′ end to the 5′ end of SEQ ID NO: 15 by four iSpC3spacer units) to form a hairpin loop MuA substrate. The DNA substratedesigns of the two MuA substrates formed are shown in FIG. 4 (a).

TABLE 5 Y- Final Reagent shaped Hairpin Concentrations Water 12 uL  14uL  0.5M NaCl 2 uL 2 uL 50 mM 0.1M Tris pH 7.5 2 uL 2 uL 10 mM SEQ IDNO: 14 and 15 (where SEQ 2 uL 10 uM ID NO: 14 is attached at its 3′ endto the 5′ end of SEQ ID NO: 15 by four iSpC3 spacer units) (100 uM) SEQID NO: 19 and 9 (where SEQ 2 uL 10 uM ID NO: 19 is attached at its 3′end to the 5′ end of SEQ ID NO: 9 by four iSpC3 spacer units) (100 uM)SEQ ID NO: 19 and 20 (where SEQ 2 uL 10 uM ID NO: 19 is attached at its3′ end to the 5′ end of SEQ ID NO: 15 by four iSpC3 spacer units) (100uM) Total 20 uL  20 uL 

2.2 Fragmentation of the DNA Template Using the MuA Transposase

Double-stranded Lambda DNA (SEQ ID NO: 13 corresponds to the sequence ofthe sense strand) was fragmented into approximately 5-10 kB lengthstrands using a MuA transposase. The MuA transposase inserted the MuAsubstrates (the Y-shaped and the hairpin MuA substrates) which wereannealed in section 3.1. The sample was prepared as shown in Table 6below. The sample was then incubated at 30° C. for 1 hour and heatinactivated at 75° C. for 10 minutes. The sample was then furtherpurified using a QIAquick™ PCR Purification kit (Qiagen) and eluted in26 μL.

TABLE 6 Sample Final Reagent Volume 1 Concentrations Water 17.7 uL  Lambda DNA (90 ng/uL) (SEQ ID NO: 22.3 uL    2 μg 13 shows the sensestrand sequence only) Y-shaped MuA substrate (1 uM) 8 uL 100 nM HairpinMuA substrate (1 uM) 8 uL 100 nM 5x Buffer (125 mM Tris (pH 8.0), 16 uL 1x 50 mM MgCl₂, 550 mM NaCl, 0.25% Triton X-100 and 50% Glycerol) MuA (4uM, Thermo, catalogue No. F- 8 uL 400 nM 750C) Total 80 uL 2.3 USER Digest of Fragmented Lambda DNA with Inserted MuA Substrates

Purified sample volume 1 from step 3.2 was then treated with USER™digest in order to remove the dUMP from SEQ ID NOs: 19. See Table 7below for appropriate volumes and concentrations. The sample was thenincubated at 37° C. for 30 minutes before it was cooled in an ice block.

TABLE 7 Final Reagent Sample Volume 2 Concentrations Sample Volume 1 26uL 8 pmol of U 10x DNA ligase buffer  3 uL 1x USER (1 U/uL)  1 uL 1 UTotal 30 uL

2.4 Repair of Single-Stranded Gap in the Double-Stranded Lambda DNAConstruct Fragments

Sample Volume 2 produced after treatment with USER™ was then treatedwith DNA polymerase and ligase in order to close the single-strandedgap. Sample volume 3 (see table 8 below for appropriate volumes andconcentrations) was incubated for 30 minutes at 16° C. and then EDTA(0.5 M, 10 μL) was added to sample volume 3. A QIAquick™ PCRPurification kit was then used to purify each sample, which was elutedin 50 μL of water. An aliquot of the purified sample (1 μL) was run onan Agilent 12000 chip to quantify the sample and Tris-HCl and NaCl (pH7.5) until were added to the rest of the sample until the concentrationswere 10 mM and 50 mM respectively. Finally, SEQ ID NO: 16 (3′ end of thesequence has six iSp18 spacers attached to two thymine residues and a 3′cholesterol TEG, 0.5 μM) was annealed to the purified sample.

TABLE 8 Reagent Sample Volume 3 Final Concentrations Water 6.2 uL  Sample Volume 2 30 uL  10x DNA ligase buffer 1 uL 1x dNTPs (10 mM) 0.8uL   200 uM T4 DNAP exo(□) 1 uL (Lucigen) Ligase (NEB; M0202M) 1 uL 1xTotal 40 uL 

2.5 Electrophysiology Experiment Showing Helicase Controlled DNAMovement of the Purified and Fragmented Lambda DNA Construct

Prior to setting up the experiment, the Lambda DNA construct (0.2 nM,5-10 kB fragments of Lambda DNA which have had the Y-shaped MuAsubstrates and the hairpin MuA substrates attached to either end of thefragments by the MuA transposase (see FIG. 4 (b) for an exampleconstruct)) and Trwc Cba (SEQ ID NO: 9, 1 μM) were pre-incubatedtogether for 1 hour in buffer (50 mM CAPS, pH 10.0 (pH altered to pH10.0 by addition of NaOH), 100 mM NaCl).

Electrical measurements were acquired from single MspA nanopores(MapA-B2C) inserted in block co-polymer in buffer (600 mM KCl, 25 mMKH2PO4, 75 mM Potassium Ferrocyanide (II), 25 mM Potassium ferricyanide(III), pH 8). After achieving a single pore in the bilayer, then buffer(1 mL, 600 mM KCl, 25 mM KH2PO4, 75 mM Potassium Ferrocyanide (II), 25mM Potassium ferricyanide (III), pH 8) was flowed through the system toremove any excess MspA nanopores (MspA-B2C) and the experimental systemwas placed on a cooler plate set to 8° C. which gave a systemtemperature of ˜15° C. MgCl₂ (10 mM) and dTTP (5 mM) were mixed togetherwith buffer (600 mM KCl, 25 mM KH2PO4, 75 mM Potassium Ferrocyanide(II), 25 mM Potassium ferricyanide (III), pH 8) and then added to theLambda DNA construct (0.2 nM), Trwc Cba (SEQ ID NO: 9, 1 μM) buffer (50mM CAPS, pH 10.0 (pH altered to pH 10.0 by addition of NaOH), 100 mMNaCl) pre-mix. The pre-mix was then added to the single nanoporeexperimental system. Experiments were carried out for two hoursfollowing a potential flip process (+120 mV for 30 mins, then −100 mVfor 2 seconds and then 0 mV for 2 seconds) and helicase-controlled DNAmovement was monitored.

2.6 Results and Discussion

Helicase controlled DNA movement was observed for the Lambda DNAconstruct, an example of a helicase-controlled DNA movement is shown inFIG. 5. The iSpC3 spacers present in the Lambda DNA construct produced acharacteristic block level highlighted by the numbers 1, 2 and 3 in FIG.5. The Y-shaped MuA substrate has four iSpC3 spacers in either strandand the hairpin MuA substrate also has four iSpC3 spacers, each iSpC3spacer allows more current to flow as that region of the Lambda DNAconstruct translocates through the nanopore. If the sample preparationhas occurred successfully then the iSpC3 spacer events will be observedat the beginning of the Lambda DNA construct, in the middle (marking thetransition between sense and antisense sequences) and at the end. FIG. 5clearly shows three instances of increased current flow which correspondto the iSpC3 spacer regions. Therefore, the sample preparation procedureeffectively introduced MuA substrates into the Lambda DNA to produce theLambda DNA constructs shown in FIG. 4 (b). Upon capture of the DNA bythe nanopore, the enzyme (labelled A) is moved past the dsDNA region(where the tether hybridises to the construct) and the spacers by theforce of the applied potential across the nanopore.

Example 3

The DNA construct used in this example was produced by fragmentation ofLambda DNA into ˜5-10 kB fragments using MuA. This example is similar tothe one described in Example 2, however, the sample preparationprocedure is different (steps 2.3 and 2.4 as described above are notrequired) as the transposase sequences contain inosines in this example.The enzyme was moved past the dsDNA region and the spacers by the forceof the applied potential across the nanopore.

Materials and Methods 3.1 Anneal of DNA Strands to Form Y-Shaped andHairpin MuA Substrates

The Y-shaped 2 and hairpin 2 MuA substrates were prepared as describedin Example 2.1 above. Volumes, concentrations and sequences that wereused in this example are detailed in table 9 below. The DNA substratedesigns of the two constructs formed are shown in FIG. 6 (a).

TABLE 9 Y- Final Reagent shaped 2 Hairpin 2 Concentrations Water 12 uL 14 uL  0.5M NaCl 2 uL 2 uL 50 mM 0.1M Tris pH 7.5 2 uL 2 uL 10 mM SEQ IDNO: 14 and 15 (where 2 uL 10 uM SEQ ID NO: 14 is attached at its 3′ endto the 5′ end of SEQ ID NO: 15 by four iSpC3 spacer units) (100 uM) SEQID NO: 18 and 9 (where 2 uL 10 uM SEQ ID NO: 18 is attached at its 3′end to the 5′ end of SEQ ID NO: 9 by four iSpC3 spacer units) (100 uM)SEQ ID NO: 18 and 15 (where 2 uL 10 uM SEQ ID NO: 18 is attached at its3′ end to the 5′ end of SEQ ID NO: 15 by four iSpC3 pacer units) (100uM) Total 20 uL  20 uL 

3.2 Fragmentation of the DNA Template Using the MuA Transposase

Double-stranded Lambda DNA (SEQ ID NO: 13 shows the sequence of thesense strand only) was fragmented into approximately 5-10 kB lengthstrands using a MuA transposase. The MuA transposase inserted the MuAsubstrates (the Y-shaped 2 and the hairpin 2 MuA substrates) which wereannealed in section 3.1. The sample was prepared by an analogousprocedure as that described in Section 2.2 and table 6 above except theMuA substrates used were the Y-shaped 2 and the hairpin 2 MuAsubstrates. In this case the purified sample X was eluted in a volume of20 μL.

3.3 Nick Repair in the Double-Stranded Lambda DNA Construct Fragments

Once the Y-shaped 2 and the hairpin 2 MuA substrates have been insertedinto the fragmented Lambda DNA it is necessary to repair the nick in thestrand and join the inosines to the Lambda DNA fragment to produce acomplete double-stranded Lambda DNA fragment. One reaction was assembledon ice as described in Table 10 below. The sample was incubated at 16°C. for 60 mins before EDTA (10 μL, 0.5 M) was added to the sample. Theresultant sample mixture was purified using a QiaQuick™ purify and waseluted in 50 μL of water. An aliquot of the purified sample (1 μL) wasrun on an Agilent 12000 chip to quantify the sample and Tris-HCl andNaCl (pH 7.5) were added to the rest of the sample until theconcentrations were 10 mM and 50 mM respectively. Finally, SEQ ID NO: 16(3′ end of the sequence has six iSp18 spacers attached to two thymineresidues and a 3′ cholesterol TEG, 0.5 μM) was annealed to the purifiedLambda DNA construct.

TABLE 10 Reagent Sample Z Final Concentrations Sample X 16 uL 2x DNAligase buffer 20 uL 1x Ligase (NEB; M0202M)  4 uL Total 40 uL

3.4 Electrophysiology Experiment Showing Helicase Controlled DNAMovement of the Purified and Fragmented Lambda DNA Construct

Prior to setting up the experiment, the Lambda DNA construct (0.2 nM,5-10 kB fragments of Lambda DNA which have had the Y-shaped 2 and thehairpin 2 MuA substrates attached to either end of the fragments by theMuA transposase (see FIG. 6 (b) for an example construct)) and Trwc Cba(SEQ ID NO: 17, 1 μM) were pro-incubated together for 1 hour in buffer(50 mM CAPS, pH 10.0 (pH altered to pH 10.0 by addition of NaOH), 100 mMNaCl).

Electrical measurements were acquired from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer (600 mM KCl, 25 mMKH2PO4, 75 mM Potassium Ferrocyanide (II), 25 mM Potassium ferricyanide(III), pH 8). After achieving a single pore in the bilayer, then buffer(3 mL, 600 mM KCl, 25 mM KH2PO4, 75 mM Potassium Ferrocyanide (II), 25mM Potassium ferricyanide (III), pH 8) was flowed through the system toremove any excess MspA nanopores (MspA-B2C) and the experimental systemwas placed on a cooler plate set to 8° C. which gave a systemtemperature of ˜15° C. MgCl₂ (10 mM) and dTTP (5 mM) were mixed togetherwith buffer (600 mM KCl, 25 mM KH2PO4, 75 mM Potassium Ferrocyanide(II), 25 mM Potassium ferricyanide (III), pH 8) and then added to theLambda DNA construct (0.2 nM), Trwc Cba (SEQ ID NO: 17, 1 μM) buffer (50mM CAPS, pH 10.0 (pH altered to pH 10.0 by addition of NaOH), 100 mMNaCl) pre-mix. The pre-mix was then added to the single nanoporeexperimental system. Experiments were carried out for two hoursfollowing a potential flip process (+120 mV for 30 mins, then −100 mVfor 2 seconds and then 0 mV for 2 seconds) and helicase-controlled DNAmovement was monitored.

3.5 Results and Discussion

Helicase controlled DNA movement was observed for the Lambda DNAconstruct, an example of a helicase-controlled DNA movement is shown inFIG. 7. The iSpC3 spacers present in the Lambda DNA construct produced acharacteristic block level highlighted by numbers 1-3 in FIG. 7. TheY-shaped 2 MuA substrate has four iSpC3 spacers in either strand and thehairpin 2 MuA substrate also has four iSpC3 spacers, each iSpC3 spacerallows more current to flow as that region of the Lambda DNA constructtranslocates through the nanopore. If the sample preparation occurredsuccessfully then the iSpC3 spacer events will be observed at thebeginning of the Lambda DNA construct, in the middle (making thetransition between the sense and antisense sequences) and at the end.FIG. 7 clearly shows three instances of increased current flow whichcorrespond to the iSpC3 spacer regions. Therefore, the samplepreparation procedure effectively introduced MuA substrates into theLambda DNA to produce the Lambda DNA constructs shown in FIG. 6 (b).Upon capture of the DNA by the nanopore, the enzyme (labelled A) is pastthe dsDNA region (where the tether hybridises to the construct) and thespacers by the force of the applied potential across the nanopore. Owingto the use of the inosines in the Y-shaped 2 and the hairpin 2 MuAsubstrates, the steps in the sample preparation procedure were reducedas once the MuA substrates had been inserted all that was necessary wasto close the nicks in the double-stranded DNA constructs.

Example 4

This Example compares the ability of a TrwC Cba monomer (SEQ ID NO: 17),to control the movement of intact DNA strands (attached to the 5′ end ofSEQ ID NO: 23 is 28 iSpC3 spacers units the last of which has anadditional two T's attached to the 5′ end of the spacer group, attachedto the 3′ end of SEQ ID NO: 23 is a further four iSpC3 spacers which areattached to the 5′ end of SEQ ID NO: 24, where SEQ ID NO: 12 ishybridised to a region of SEQ ID NO: 23) through a nanopore, to that ofthe TrwC Cba Q276C-3.4 kDa dimer (where each monomer unit comprises SEQID NO: 17 with the mutation Q276C, with one monomer unit being linked tothe other via position 276 of each monomer unit using a 3.4 kDa PEGlinker). The DNA construct used in this example is shown in FIG. 22 (thehelicase is capable of binding to the region labelled B). When the DNAconstruct is captured by the nanopore, the applied potential across thenanopore moves the enzyme past the dsDNA region (where the tetherhybridises to the construct) and spacers and helicase controlled DNAmovement is observed.

Upon comparison of the helicase controlled movement of the monomer withthe dimer, it was observed that the dimer resulted in a greaterpercentage of long dwell helicase-controlled DNA movement (a long dwellmovement is a helicase-controlled DNA movement which is more than threestandard deviations away from the mean of the major population ofhelicase-controlled DNA movements) than the monomer.

Materials and Methods

Prior to setting up the experiment, the DNA (1 nM, attached to the 5′end of SEQ ID NO: 23 is 28 iSpC3 spacers units the last of which has anadditional two T's attached to the 5′ end of the spacer group, attachedto the 3′ end of SEQ ID NO: 23 is a further four iSpC3 spacers which areattached to the 5′ end of SEQ ID NO: 24, where SEQ ID NO: 12 ishybridised to a region of SEQ ID NO: 23) and the enzyme (either a TrwCCba monomer (1 nM, SEQ ID NO: 17) or TrwC Cba Q276C-3.4 kDa dimer (0.3nM, where each monomer unit comprises SEQ ID NO: 17 with the mutationQ276C, with one monomer unit being linked to the other via position 276of each monomer unit using a 3.4 kDa PEG linker)) were pre-incubatedtogether for >16 hours.

Electrical measurements were acquired from single MspA nanoporesMS(G75S/G77S/L88N/Q126R)8 MspA (SEQ ID NO: 2 with the mutationsG75S/G77S/L88N/Q126R) inserted in block copolymer in buffer (625 mM KCl,100 mM Hepes, 75 mM Potassium Ferrocyanide (II), 25 mM Potassiumferricyanide (III), pH 8). MgCl₂ (10 mM) and dTTP (5 mM) were mixedtogether with buffer (625 mM KCl, 100 mM Hepes, 75 mM PotassiumFerrocyanide (II), 25 mM Potassium ferricyanide (III), pH 8) and thenadded to the DNA (construct described previously), enzyme pre-mix(either a TrwC Cba monomer (1 nM, SEQ ID NO: 17) or TrwC Cba Q276C-3.4kDa dimer (I nM, where each monomer unit comprises SEQ ID NO: 17 withthe mutation Q276C, with one monomer unit being linked to the other viaposition 276 of each monomer unit using a 3.4 kDa PEG linker)). Afterachieving a single pore in the bilayer, the pre-mix was added to thesingle nanopore experimental system. Experiments were carried out at aconstant potential of +120 mV and helicase-controlled DNA movement wasmonitored.

Results and Discussion

Helicase controlled DNA movement was observed for the helicase TrwC Cbamonomer (SEQ ID NO: 17) and TrwC Cba Q276C-3.4 kDa dimer (where eachmonomer unit comprises SEQ ID NO: 17 with the mutation Q276C, with onemonomer unit being linked to the other via position 276 of each monomerunit using a 3.4 kDa PEG linker). Upon capture of the DNA construct bythe nanopore the helicase was moved past the dsDNA region (where thetether hybridises to the construct) and the spacers and helicasecontrolled movement was observed.

Of the helicase-controlled DNA movements observed there is a majorpopulation which accounts for around 95% of movements detected, however,there is a small percentage of movements which are significantly longerin dwell time (more than three standard deviations away from the mean ofthe major population of helicase-controlled DNA movements). These longermovements allow improved data analysis. When the TrwC Cba Q276C-3.4 kDadimer (1 nM) was used to control DNA movement then a much higherpercentage (20% for the TrwC Cba Q276C-3.4 kDa dimer in comparison toand 5% for the TrwC Cba monomer) of these longer dwell time movements(more than three standard deviations away from the mean of the majorpopulation of helicase-controlled DNA movements) was observed. The useof the dimer helicase provides an advantage over the monomer as itallows improved data analysis in the nanopore sequencing system.

Example 5

This Example illustrates that Sp9 spacer units can be used to stall themovement of Hel308 Mbu (SEQ ID NO: 28) (when provided with both ATP andMgCl2) in a fluorescence based assay for testing enzyme activity.

Materials and Methods

Three different custom fluorescent substrates (A=(control strandscontaining no spacers) SEQ ID NOs: 25 and 26, B=(strand containing asingle Sp9 spacer) SEQ ID NO: 27 attached at its 3′ end by one Sp9spacers to the 5′ end of SEQ ID NO: 29 and hybridised to SEQ ID NO: 26,C=(strand containing four Sp9) SEQ ID NO: 27 attached at its 3′ end byfour Sp9 spacers to the 5′ end of SEQ ID NO: 29 and hybridised to SEQ IDNO: 26) were used to assay the ability of Hel308 Mbu (SEQ ID NO: 28) todisplace hybridised dsDNA. FAM labelled DNA (for fluorescent substrateA=SEQ ID NO: 25, B=SEQ ID NO: 27 attached by its 3′ end to one sp9spacer which is attached to the 3′ end of SEQ ID NO: 29, C=SEQ ID NO: 27attached by its 3′ end to four sp9 spacers which are attached to the 3′end of SEQ ID NO: 29) is annealed to the partially complementary strandwhich has a black-hole quencher attached to its 3′ end (SEQ ID NO: 26)in a one to one ratio (1 uM of each strand) in 400 mM KCl, 100 mM HEPESpH8, 10 mM MgCl₂, 1 mg/ml BSA. The strands were annealed at roomtemperature for 30 minutes. The annealed DNA (A=SEQ ID NOs: 25 and 26,B=SEQ ID NO: 27 attached at its 3′ end by one Sp9 spacers to the 5′ endof SEQ ID NO: 29 and hybridised to SEQ ID NO: 26, C=SEQ ID NO: 27attached at its 3′ end by four Sp9 spacers to the 5′ end of SEQ ID NO:29 and hybridised to SEQ ID NO: 26) was diluted to 50 nM in 400 mM KCl,100 mM HEPES pH8, 10 mM MgCl₂, 1 mg/ml BSA, 1 mM ATP (1 uM capture DNA(SEQ ID NO: 27 also present). A sample of Hel308 Mbu (SEQ ID NO: 28) wasdiluted to 475 nM in 400 mM KCl, 100 mM HEPES pH8, 10 mM MgCl2, 1 mg/mlBSA. Hel308 Mbu (12 nM) was then assayed (as described below and shownin FIGS. 8 and 9) against 48.75 nM annealed DNA in 400 mM KCl, 100 mMHEPES pH8, 10 mM MgCl₂, 1 mg/ml BSA, 0.975 mM ATP (0.975 uM capture DNAalso present).

The control strand A is shown in FIG. 8, where in 1A) the fluorescentsubstrate strand (48.75 nM final) has a 3′ ssDNA overhang, and a 40 basesection of hybridised dsDNA. The upper strand, containing the 3′ ssDNAoverhang, has a carboxyfluorescein base attached to the 5′ end of SEQ IDNO: 25, and the hybrised complement has a black-hole quencher (BHQ-1)base attached to the 3′ end of SEQ ID NO: 26. When hybridised thefluorescence from the fluorescein is quenched by the local BHQ-1, andthe substrate is essentially non-fluorescent. 1 μM of a capture strand(SEQ ID NO: 27) that is part-complementary to the lower strand of thefluorescent substrate is included in the assay. As shown in 2A), in thepresence of ATP (0.975 mM) and MgCl₂ (10 mM), helicase (12 nM) added tothe substrate binds to the 3′ tail of the fluorescent substrate, movesalong the upper strand, and displaces the complementary strand. As shownin 3A), once the complementary strand with BHQ-1 is fully displaced thefluorescein on the major strand fluoresces. As shown in 4A), thedisplaced strand preferentially anneals to an excess of capture strandto prevent re-annealing of initial substrate and loss of fluorescence.FIG. 9 shows the steps of the assay for strands B (1B-3B) and C (1C-3C),for these strands the Sp9 spacers stall the helicase and prevent it fromseparating the strand with the fluorescein attached from the strand withthe black hole quencher attached.

Results and Discussion

The graph in FIG. 10 shows the initial rate of activity in buffersolution (100 mM Hepes pH8.0, 0.975 mM ATP, 10 mM MgCl₂, 1 mg/ml BSA,48.75 nM fluorescent substrate DNA (substrates A, B and C as discussedabove), 0.975 μM capture DNA (SEQ ID NO: 27)) for the Hel308 Mbu(labeled A in FIGS. 8 and 9; SEQ ID NO: 28) at 400 mM of KCl. At thesalt concentration investigated the Hel308 Mbu (SEQ ID NO: 28) exhibiteddsDNA turnover of the control strand A. However, FIG. 10 clearlyindicates that for both constructs which have Sp9 spacers in thesequence (B (one Sp9 spacer) and C (four Sp9 spacers)) the dsDNAturnover was abolished. This indicates that in the presence of ATP andMgCl2 the Sp9 spacers stall the Hel308 Mbu enzyme in free solution.

Example 6

This Example illustrates that idSp groups can be used to stall themovement of Hel308 Mbu (SEQ ID NO: 28) (when provided with both ATP andMgCl2) in a fluorescence based assay for testing enzyme activity.

Materials and Methods

Four different custom fluorescent substrates (D=(control strandcontaining no spacers) SEQ ID NOs: 32 and 26, E=(strand containing asingle idSp spacer) SEQ ID NO: 27 attached at its 3′ end by one idSpgroup to the 5′ end of SEQ ID NO: 30 and hybridised to SEQ ID NO: 26,F=(strand containing four idSp) SEQ ID NO: 27 attached at its 3′ end byfour idSp groups to the 5′ end of SEQ ID NO: 31 and hybridised to SEQ IDNO: 26 and G=(second control strand containing no spacers) SEQ ID NOs:33 and 26) were used to assay the ability of Hel308 Mbu (SEQ ID NO: 28)to displace hybridised dsDNA. FAM labelled DNA (for fluorescentsubstrate D=SEQ ID NO: 32, E=SEQ ID NO: 27 attached by its 3′ end to oneidSp group which is attached to the 3′ end of SEQ ID NO: 30, F=SEQ IDNO: 27 attached by its 3′ end to four idSp groups which are attached tothe 3′ end of SEQ ID NO: 31 and G=SEQ ID NO: 33) is annealed to thepartially complementary strand which has a black-hole quencher attachedto its 3′ end (SEQ ID NO: 26) in a 1 to 1.2 ratio (1:1.2 μM) in 400 mMKCl, 100 mM HEPES pH8, 10 mM MgCl₂, 1 mg/ml BSA. The strands wereannealed at room temperature for 15 minutes. The annealed DNA (D=SEQ IDNOs: 32 and 26, E=SEQ ID NO: 27 attached at its 3′ end by one idSp groupto the 5′ end of SEQ ID NO: 30 and hybridised to SEQ ID NO: 26, F=SEQ IDNO: 27 attached at its 3′ end by four idSp groups to the 5′ end of SEQID NO: 31 and hybridised to SEQ ID NO: 26 and G=SEQ ID NOs: 33 and 26)was diluted to 50 nM in 400 mM KCl, 100 mM HEPES pH8, 10 mM MgCl₂, 1mg/ml BSA, 1 mM ATP (1 uM capture DNA (SEQ ID NO: 27 also present).Hel308 Mbu (12 nM) was then assayed (as described previously in Example5 (except the DNA constructs are different and contain idSp groupsinstead of Sp9 spacers) and shown in FIGS. 8 and 9 (again where the Sp9groups in these figures are replaced with idSp groups)) against 48.75 nMannealed DNA in 400 mM KCl, 100 mM HEPES pH8, 10 mM MgCl₂, 1 mg/ml BSA,0.975 mM ATP (0.975 uM capture DNA also present).

Results and Discussion

The graph in FIG. 11 shows the initial rate of activity in buffersolution (100 mM Hepes pH 8.0, 0.975 mM ATP, 10 mM MgCl₂, 1 mg/ml BSA,48.75 nM fluorescent substrate DNA (substrates D, E, F and G asdiscussed above), 0.975 μM capture DNA (SEQ ID NO: 27)) for the Hel308Mbu (labelled A in FIGS. 8 and 9; SEQ ID NO: 28) at 400 mM of KCl. Atthe salt concentration investigated the Hel308 Mbu (SEQ ID NO: 28)exhibited dsDNA turnover of the control strands D and G. However, FIG.11 clearly indicates that for the construct which has four idSp spacersin the sequence (F) the dsDNA turnover was abolished. This indicatesthat in the presence of ATP and MgCl2 the four idSp groups stall theHel308 Mbu enzyme in free solution.

Example 7

This Example illustrates a gel based assay that was used to measure theability of iSpC3 spacers and iSp18 spacers to stall the movement of T4Dda-E94C/A360C.

Materials and Methods

The annealed DNA complexes (sequences tested are shown in table 11below) were mixed in a ratio of (1:1, v/v) with T4 Dda-E94C/A360C in 25mM phosphate pH 8.0, 200 mM KCl giving final concentrations of T4Dda-E94C/A360C (2000 nM) and DNA (100 nM). The helicase was allowed tobind to the DNA for 2 hours at ambient temperature. Capture strand (SEQID NO: 37, 20 μM) was added to each sample to bind any unbound enzymeand the samples incubated at ambient temperature for 30 mins. Buffer wasadded to the samples (DNA construct from table 11=50 nM, capture DNA(SEQ ID NO: 37)=10 μM and T4 Dda-E94C/A360C=1000 nM) and they wereincubated at ambient temperature for one hour (either Buffer 1=25 mMphosphate pII 8.0, 200 mM KCl, 20 mM MgCl2, 10 mM ATP or Buffer 2=25 mMphosphate pH 8.0, 1 M KCl, 25 mM potassium ferricyanide(III), 75 mMpotassium ferrocyanide, 20 mM MgCl2, 10 mM ATP). Loading buffer (25 mMPhosphate pH8.0, 151.5 mM KCl, 25% Glycerol, 125 mM EDTA) is added toeach sample to quench the helicase activity. The samples were loadedonto 4-20% TBE gel and the gel run at 160 V for 1.5 hours. The Gel wasthen stained with SYBR gold in order to observe the DNA bands.

TABLE 11 DNA construct Number DNA sequences which make up the construct1 (positive control, no SEQ ID NO: 34 hybridised to SEQ ID NO: 35spacers) 2 (4 × iSpC3 spacers) SEQ ID NO: 9 attached at its 3′ end tofour iSpC3 spacers which are attached to the 5′ end of SEQ ID NO: 36hybridised to SEQ ID NO: 35 3 (8 × iSpC3 spacers) SEQ ID NO: 9 attachedat its 3′ end to eight iSpC3 spacers which are attached to the 5′ end ofSEQ ID NO: 36 hybridised to SEQ ID NO: 35 4 (12 × iSpC3 spacers) SEQ IDNO: 9 attached at its 3′ end to twelve iSpC3 spacers which are attachedto the 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35 5 (1 × iSp18spacer) SEQ ID NO: 9 attached at its 3′ end to one iSp18 spacer whichare attached to the 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 356 (2 × iSp18 Spacer) SEQ ID NO: 9 attached at its 3′ end to two iSp18spacers which are attached to the 5′ end of SEQ ID NO: 36 hybridised toSEQ ID NO: 35 7 (3 × iSp18 spacer) SEQ ID NO: 9 attached at its 3′ endto three iSp18 spacers which are attached to the 5′ end of SEQ ID NO: 36hybridised to SEQ ID NO: 35 8 (4 × iSp18 spacer) SEQ ID NO: 9 attachedat its 3′ end to four iSp18 spacers which are attached to the 5′ end ofSEQ ID NO: 36 hybridised to SEQ ID NO: 35 9 (5 × iSp18 spacer) SEQ IDNO: 9 attached at its 3′ end to five iSp18 spacers which are attached tothe 5′ end of SEQ ID NO: 36 hybridised to SEQ ID NO: 35

Results and Discussion

FIG. 12 show the gel of the positive control experiment where the DNAconstruct contains no spacers to stall the helicase (T4 Dda-E94C/A360C).Lane 2 of FIG. 14 shows that the up to 5 helicase enzymes can bind ontothe single-stranded section of SEQ ID NO: 34. Upon the addition of ATPand MgCl2, the higher bands corresponding to the dsDNA construct boundto multiple enzymes disappears and band corresponding to the ssDNAconstruct (labelled X and corresponding to SEQ ID NO: 34 only) increasesin intensity. This indicates that when the helicase is provided withfuel it moves along the DNA and displaces the hybridised complementarystrand (SEQ ID NO: 35).

FIG. 15 shows an example of a gel showing the enzyme activity experimentfor DNA constructs 7-9 of table 11 (e.g. DNA which has three, four orfive iSp18 spacers just before the ssDNA/dsDNA junction). Lanes 1-4correspond to three iSp18 spacers, lanes 5-7 correspond to four iSp18spacers and lanes 9-12 correspond to five iSp18 spacers. Lanes 2, 6 and10 show that, similarly to the control, up to five helicase enzymes canbind onto the single-stranded section of the DNA constructs 7-9 in table11. Upon the addition of ATP and MgCl2 the helicases will be providedwith the necessary components in order to move along the DNA strand.Lanes 3-4, 7-8 and 11-12 show the various DNA constructs under twodifferent buffer conditions (buffer 1 and 2) after the addition of ATPand MgCl2. Two bands are clearly visible at the region labelled 1Y and1X. 1Y corresponds to the dsDNA construct with one helicase stalled andbound. This shows that three, four and five iSP18 spacers are able tostall the helicase under the conditions tested. 1X corresponds to thessDNA construct with one helicase stalled and bound. This band hasresulted from multiple helicases binding to the DNA construct and thehelicases pushing the front helicase past the stalling groups, thusdisplacing the short complementary strand (SEQ ID NO: 35). However, thisband still has one helicase bound as a single helicase cannot move pastthe iSp18 spacers (e.g. species E in FIG. 13 results). In the case of 4and 5 iSp18 spacers being used to stall the helicase there are faintbands at level 2Y. This shows that under the conditions tested, when 4/5iSp18 spacers are used it is possible to stall the movement of up to twohelicases.

Of the other spacer combinations investigated (entries 2-6 of table 11)in at least one of the buffer conditions tested both iSpC3 and iSp18spacers were capable of stalling one helicase. Of the two bufferconditions tested, generally more efficient stalling was observed forbuffer 2 than buffer 1. The greater the number of spacers included themore efficient the stalling of the helicases under the conditionstested.

Example 8

This Example investigates the number of bases needed to control thebinding of only one or two T4 Dda-E94C/A360C helicases in a particularregion.

Materials and Methods

DNA constructs (1 μM or 100 nM final concentration) detailed below inTable 12 were incubated in appropriate buffer with serially diluted T4Dda-E94C/A360C. The samples were then loaded on 4-20% TBE gels and runat 160 V for 90 minutes. The gels containing entries 1-6 were thenstained using SYBR. FIG. 16 shows the type of DNA construct used in thisexperiment. The length of the region labelled 1 is varied from 2 to 50to optimise conditions so that it is possible to control the number ofT4 Dda-E94C/A360C helicases which can bind to the DNA.

TABLE 12 Concentrations of Incubation T4 Dda - Entry DNA ConstructBuffer Conditions E94C/A360C 1 1 μM, Five iSpC3 spacers attached 25 mMovernight at 5000 nM, 2500 nM, to the 5′ end of SEQ ID NO: 37,phosphate, room 1250 nM, 625 nM, which is attached at its 3′ end to151.5 mM temperature 312.5 nM, 0M four iSpC3 spacers which are KCl pHattached to the 5′ end of SEQ ID 8.0 NO: 36 which is hybridised to SEQID NO: 35. 2 1 μM, Five iSpC3 spacers attached 25 mM overnight at 5000nM, 2500 nM, to the 5′ end of SEQ ID NO: 38, phosphate, room 1250 nM,625 nM, which is attached at its 3′ end to 151.5 mM temperature 312.5nM, 0M four iSpC3 spacers which are KCl pH attached to the 5′ end of SEQID 8.0 NO: 36 which is hybridised to SEQ ID NO: 35. 3 1 μM, Five iSpC3spacers attached 25 mM overnight at 5000 nM, 2500 nM, to the 5′ end ofSEQ ID NO: 9, phosphate, room 1250 nM, 625 nM, which is attached at its3′ end to 151.5 mM temperature 312.5 nM, 0M four iSpC3 spacers which areKCl pH8.0 attached to the 5′ end of SEQ ID NO: 36 which is hybridised toSEQ ID NO: 35. 4 1 μM, Five iSpC3 spacers attached 25 mM 1.5 hours at3750 nM, 1875 nM, to the 5′ end of two thymines, phosphate, room 938 nM,469 nM, which are attached at the 3′ end to 151.5 mM temperature 235 nM,0M four iSpC3 spacers which are KCl pH attached to the 5′ end of SEQ ID8.0 NO: 36 which is hybridised to SEQ ID NO: 35. 5 1 μM, Five iSpC3spacers attached 25 mM 1.5 hours at 3750 nM, 1875 nM, to the 5′ end offour thymines, phosphate, room 938 nM, 469 nM, which are attached at the3′ end to 151.5 mM temperature 235 nM, 0M four iSpC3 spacers which areKCl pH attached to the 5′ end of SEQ ID 8.0 NO: 36 which is hybridisedto SEQ ID NO: 35. 6 1 μM, Five iSpC3 spacers attached 25 mM 1.5 hours at3750 nM, 1875 nM, to the 5′ end of eight thymines, phosphate, room 938nM, 469 nM, which are attached at the 3′ end to 151.5 mM temperature 235nM, 0M four iSpC3 spacers which are KCl pH attached to the 5′ end of SEQID 8.0 NO: 36 which is hybridised to SEQ ID NO: 35. 7 100 nM, Five iSpC3spacers 25 mM Overnight 2700 nM, 1350 nM, attached to the 5′ end of SEQID phosphate, at room 675 nM, NO: 39, which is attached at its 3′ 151.5mM temperature 337.5 nM, 169 nM, end to four iSp18 spacers which are KClpH8.0 0M attached to the 5′ end of SEQ ID NO: 36 which is hybridised toSEQ ID NO: 35. 8 100 nM, Five iSpC3 spacers 25 mM Overnight 2700 nM,1350 nM, attached to the 5′ end of SEQ ID phosphate, at room 675 nM, 338nM, NO: 40, which is attached at its 3′ 151.5 mM temperature 169 nM, 0Mend to four iSp18 spacers which are KCl pH attached to the 5′ end of SEQID 8.0 NO: 36 which is hybridised to SEQ ID NO: 35.

Results and Discussion

Each of the DNA constructs listed in Table 12 were investigated todetermine how many enzymes can bind to region 1 (shown in FIG. 16) whichhas been varied from 2 bases to 50 bases. For each of the DNA constructstested only a single band for unbound DNA was observed when no helicasewas added. Under the conditions investigated, construct 4 (which has abinding region of 2 thymine bases) and construct 5 (which has a bindingregion of 4 thymine bases) were not observed to allow any helicases tobind at any concentration (see table 14). Construct 6 (which has abinding region of 8 thymine bases see table 14) and construct 1 (whichhad a binding region of 10 thymine bases) was observed to bind onehelicase only (see table 13). Construct 2 which has a binding region of20 thymine bases allowed two enzymes to bind at the higherconcentrations tested and construct 3 which has a binding region of 50thymine bases allowed up to 6 enzymes to bind at the highestconcentrations tested (see FIG. 17 for the gel showing this experimentsee table 13). Constructs 1-6 used iSpC3 spacers to prevent binding ofthe helicase at the junction of the ssDNA/dsDNA region. Constructs 7 and8 used iSp18 spacers to prevent binding of the helicase at the junctionof the ssDNA/dsDNA region. Construct 7 (which has a binding region of 16thymines) and construct 8 (which has a binding region of 18 thymines)both allowed up to two helicases to bind under the conditions tested(see table 15). Therefore, binding regions greater than 8 thymine baseslong allow at least one enzyme to bind to the DNA constructs.

TABLE 13 DNA Construct Concentration of T4 Dda - E94C/A360C Entry No5000 2500 1250 625 312.5 1 One enzyme One enzyme One enzyme One enzymeOne enzyme bound only bound only bound only bound only. bound only.Faint band Unbound for unbound DNA DNA. 2 Two Two Two Two One enzymeenzymes enzymes enzymes enzymes bound only. bound. bound. bound. bound.One Unbound enzyme DNA bound. 3 (shown in Six enzymes Five Faint bandOne enzyme One enzyme FIG. 17) bound (lane 1 enzymes for two bound. Twobound. Two FIG. 17). bound (lane 2 enzymes enzymes enzymes FIG. 17).bound. Three bound. Three bound. Faint enzymes enzymes band for bound.Four bound. Faint three enzymes band for four enzymes bound, Fiveenzymes bound (lane 5 enzymes bound (lane 4 FIG. 17). bound (lane 3 FIG.17). FIG. 17).

TABLE 14 DNA Construct Concentration of T4 Dda - E94C/A360C Entry No3750 1875 938 469 235 4 Unbound Unbound Unbound Unbound Unbound DNAonly. DNA only. DNA only. DNA only. DNA only. 5 Unbound Unbound UnboundUnbound Unbound DNA only. DNA only. DNA only. DNA only. DNA only. 6 Oneenzyme One enzyme Faint band Faint band Unbound bound. Faint bound. forone for one DNA only. band for Unbound enzyme enzyme unbound DNA bound.bound. DNA Unbound Unbound DNA DNA

TABLE 15 DNA Construct Concentration of T4 Dda - E94C/A360C Entry No2700 1350 675 338 169 7 Two Two Faint band One Faint band enzymesenzymes for two enzyme for one bound. bound. enzymes bound. enzyme Onebound. One Unbound bound. enzyme enzyme DNA. Unbound bound. bound. FaintDNA Faint band for band for unbound unbound DNA DNA 8 Two Two Faint bandFaint band Unbound enzymes enzymes for two for one DNA bound. bound.enzymes enzyme only. Faint bound. One bound. band for enzyme Unbound onebound. DNA enzyme Unbound bound. DNA

Example 9

This Example investigates the concentration of T4 Dda-E94C/A360Chelicase which when added to the DNA construct X (described and shown inFIG. 18) results in the binding of two helicases.

Materials and Methods

Two DNA constructs were tested one which has a complementary strand ofDNA which is not forked (SEQ ID NO: 42 is hybridised to the DNAconstruct shown in FIG. 18) and one which is forked (SEQ ID NO: 12(which has 6 iSp18 spacers attached to the 3′ end) is hybridised to theDNA construct shown in FIG. 18). The DNA strands shown in Table 16 wereannealed at 1 μM in 25 mM phosphate pH 8.0, 1515.5 mM KCl (a 10% excessof complementary strands SEQ ID NO: 43 (in entries 9 and 10 below) andSEQ ID NO: 12 (entry 10 below) or SEQ ID NO: 42 (entry 9 below) wasused).

TABLE 16 DNA Construct Entry No: DNA Strands Annealed 9 25 SpC3 spacersare attached to the 5′ end of SEQ ID NO: 37 which is attached at its 3′end to two iSp18 spacers. The iSp18 spacers are attached to another DNAfragment of SEQ ID NO: 37 which again is attached at its 3′ end to theanother two iSp18 spacers. The second instance of iSp18 spacers isattached to the 5′ end of SEQ ID NO: 10 which is attached its 3′ end tofour nitroindoles. The four nitroindoles are then attached to the 5′ endof SEQ ID NO: 41. SEQ ID NO: 41 is hybridised to the complementarystrand SEQ ID NO: 43. SEQ ID NO: 10 is hybridised to the non-forkedcomplementary strand SEQ ID NO: 42. 10 25 SpC3 spacers are attached tothe 5′ end of SEQ ID NO: 37 which is attached at its 3′ end to two iSp18spacers. The iSp18 spacers are attached to another DNA fragment of SEQID NO: 37 which again is attached at its 3′ end to the another two iSp18spacers. The second instance of iSp18 spacers is attached to the 5′ endof SEQ ID NO: 10 which is attached its 3′ end to four nitroindoles. Thefour nitroindoles are then attached to the 5′ end of SEQ ID NO: 41. SEQID NO: 41 is hybridised to the complementary strand SEQ ID NO: 43. SEQID NO: 10 is hybridised to the forked complementary strand SEQ ID NO: 12which has 6 iSp18 spacers attached at its 3′ end.

T4 Dda-E94C/A360C was buffer exchanged into 25 mM phosphate pH 8.0,151.5 mM KCl and serially diluted. The helicase and DNA were then mixed(1:1, v/v) with the DNA construct samples 9 and 10 described above(final concentration DNA=100 nM, helicase concentrationsinvestigated=3800 nM, 1900 nM, 950 nM, 475 nM, 238 nM, 0 nM). The DNAand enzyme volumes were then incubated at ambient temperature for 1.5hours. Dye free loading buffer (5×, 7.5 μL) was added to each sample (30μL). Each sample (37.5 μL) was then loaded onto 4-20% TBE gel and run at160 V for 90 minutes. The gel was then stained using SYBR.

Results and Discussion

The two DNA constructs listed in Table 16 were investigated to determinewhat concentration of T4 Dda-E94C/A360C helicase is required in order topromote binding of two helicases. For each of the DNA constructs testedonly a single band for unbound DNA was observed when no helicase wasadded. Under the conditions investigated, both constructs 9 (non-forkedconstruct) and 10 (forked) observed binding of one helicase from 238 nMhelicase and two enzymes from 475 nM and higher. As the concentration ofenzyme was increased the band corresponding to two enzymes boundincreased in intensity. The design of the DNA construct shown in FIG. 18allows the binding of only two enzymes at concentrations as high as 3800nM. Therefore, the spacers investigated do not allow the helicase tobind to them when the constructs are pre-incubated.

Example 10

This Example shows how the T4 Dda-E94C/A360C is stalled by four iSp18spacers in free solution until the construct (DNA construct X1) iscaptured by the nanopore. Upon capture the force of the appliedpotential moves the enzyme T4 Dda-E94C/A360C past the stalling spacerand allows enzyme controlled DNA movement of the lambda constructthrough the nanopore.

Materials and Methods

Prior to setting up the experiment, the DNA construct X1 (0.13 μL, 100nM) and T4 Dda-E94C/A360C (15.6 μL, 250 nM) were pre-incubated togetherfor 1 hour at room temperature in buffer (50 mM potassium phosphate, 253mM KCl, pH 8.0).

Electrical measurements were acquired at 30° C. (by placing theexperimental system on a cooler plate) from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer (600 mM KCl, 25 mMpotassium phosphate, 75 mM Potassium Ferrocyanide (11), 25 mM Potassiumferricyanide (III), pH 8). After achieving a single pore inserted in theblock co-polymer, then buffer (1 mL, 600 mM KCl, 25 mM potassiumphosphate, 75 mM Potassium Ferrocyanide (II), 25 mM Potassiumferricyanide (III), pH 8) was flowed through the system to remove anyexcess MspA nanopores (MspA-B2C). Potassium ferricyanide (III) (200 μMfinal concentration) was added to the DNA (0.1 nM final concentration)enzyme (3 nM final concentration) pre-mix and left to incubate for oneminute before MgCl₂ (10 mM final concentration) and ATP (1 mM finalconcentration) were mixed together with buffer (1260 μL, 600 mM KCl, 25mM potassium phosphate, 75 mM Potassium Ferrocyanide (II), 25 mMPotassium ferricyanide (III), pH 8). This experimental mix was thenadded to the single nanopore experimental system. Experiments werecarried out for six hours following a potential flip process (+180 mVfor 2 s, then 0 V for 2 s, then −120 mV for 3600s (×6 repeats) appliedat the cis side) and helicase-controlled DNA movement was monitored.

Results and Discussion

The DNA construct is shown in FIG. 20. The T4 Dda-E94C/A360C is able tobind on to the region of the construct labelled A (SEQ ID NO: 9) whenpre-incubated with the DNA construct. However, the enzyme is not able tomotor past the stalling groups (four iSp18 spacers) when in freesolution (labelled as black boxes). Therefore, the enzyme is stalled atthe iSp18 spacers (labelled B in FIG. 1) until the DNA construct iscaptured by the nanopore. Once captured by the nanopore, the force ofthe applied potential moves the enzyme T4 Dda-E94C/A360C past thestalling spacer (four iSp18 spacers) and allows enzyme controlled DNAmovement of the DNA construct X1 construct through the nanopore. Anexample of a helicase-controlled DNA movement is shown in FIG. 21. Thesection labelled 1 showed when the pT region of SEQ ID NO 38translocated through the nanopore under control of the helicase. Thesection labelled 2 showed when the iSp18 spacer translocated through thenanopore under the control of the helicase. The helicase was observed tohave a defined start point (resulting from the stalling of the helicaseby the iSp18 spacers) as the helicase events observed showed the pTregion and the iSp18 signal at the beginning of the helicase controlledDNA movements.

1. A method of moving one or more stalled helicases past one or morespacers in a polynucleotide, comprising contacting (a) the one or morestalled helicases and the polynucleotide with a transmembrane pore and(b) applying a potential across the pore and thereby moving the one ormore helicases past the one or more spacers on the polynucleotide.
 2. Amethod of controlling the movement of a target polynucleotide through atransmembrane pore, comprising: (a) providing the target polynucleotidewith one or more spacers; (b) contacting the target polynucleotide withone or more helicases such that the one or more helicases stall at theone or more spacers; (c) contacting the target polynucleotide and theone or more stalled helicases with the pore; and (d) applying apotential across the pore such that the one or more helicases move pastthe one or more spacers and control the movement of the targetpolynucleotide through the pore. 3-51. (canceled)
 52. A method forcharacterising a template polynucleotide strand said method comprising:(a) incubating the template polynucleotide strand and a polynucleotidebinding protein bound thereto, in the presence of components necessaryto facilitate a movement of the polynucleotide binding protein along thetemplate polynucleotide strand, for a period of time wherein themovement of the polynucleotide binding protein along the templatepolynucleotide strand is not detected; and (b) initiating detection ofmovement of the polynucleotide binding protein along the templatepolynucleotide strand thereby characterising a portion of the templatepolynucleotide strand.